Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts

Identification and Functional Analysis of Temperate Siphoviridae Bacteriophages of Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the family Siphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52–54 predicted genes, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A. baumannii strain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only two A. baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A. baumannii

Beta diversity of stream insects differs between boreal and subtropical regions, but land use does not generally cause biotic homogenization

Previous studies have found mixed results regarding the relationship between beta diversity and latitude. In addition, by influencing local environmental heterogeneity, land use may modify spatial taxonomic and functional variability among communities causing biotic differentiation or homogenization. We tested 1) whether taxonomic and functional beta diversities among streams within watersheds differ between subtropical and boreal regions and 2) whether land use is related to taxonomic and functional beta diversities in both regions. We sampled aquatic insects in 100 subtropical (Brazil) and 100 boreal (Finland) streams across a wide gradient of land use, including agriculture and exotic planted, secondary, and native forests. We calculated beta diversity at the watershed scale (among 5 streams in each watershed). We found higher taxonomic beta diversity among subtropical than among boreal streams, whereas functional beta diversity was similar between the 2 regions. Total land use was positively correlated with taxonomic and functional beta diversity among subtropical streams, while local environmental heterogeneity was positively correlated with beta diversity among boreal streams. We suggest that different types and intensities of land use may increase among-stream heterogeneity, promoting distinct insect assemblage compositions among streams. Our findings also suggest that beta diversity patterns and their underlying determinants are highly context dependent.Peer reviewe

Antecedents and consequences of effectuation and causation in the international new venture creation process

The selection of the entry mode in an international market is of key importance for the venture. A process-based perspective on entry mode selection can add to the International Business and International Entrepreneurship literature. Framing the international market entry as an entrepreneurial process, this paper analyzes the antecedents and consequences of causation and effectuation in the entry mode selection. For the analysis, regression-based techniques were used on a sample of 65 gazelles. The results indicate that experienced entrepreneurs tend to apply effectuation rather than causation, while uncertainty does not have a systematic influence. Entrepreneurs using causation-based international new venture creation processes tend to engage in export-type entry modes, while effectuation-based international new venture creation processes do not predetermine the entry mod

Differences in Atherosclerotic Plaque Burden and Morphology Between Type 1 and 2 Diabetes as Assessed by Multislice Computed Tomography

OBJECTIVE It is unclear whether the coronary atherosclerotic plaque burden is similar in patients with type 1 and type 2 diabetes. By using multislice computed tomography (MSCT), the presence, degree, and morphology of coronary artery disease (CAD) in patients with type 1 and type 2 diabetes were compared. RESEARCH DESIGN AND METHODS Prospectively, coronary artery calcium (CAC) scoring and MSCT coronary angiography were performed in 135 asymptomatic patients (65 patients with type 1 diabetes and 70 patients with type 2 diabetes). The presence and extent of coronary atherosclerosis as well as plaque phenotype were assessed and compared between groups. RESULTS No difference was observed in average CAC score (217 +/- 530 vs. 174 +/- 361) or in the prevalence of coronary atherosclerosis (65% vs. 71%) in patients with type 1 and type 2 diabetes. However, the prevalence of obstructive atherosclerosis was higher in patients with type 2 diabetes (n = 24; 34%) compared with that in patients with type 1 diabetes (n = 11; 17%) (P = 0.02). In addition, a higher mean number of atherosclerotic and obstructive plaques was observed in patients with type 2 diabetes. In addition, the percentage of noncalcified plaques was higher in patients with type 2 (66%) versus type 1 diabetes (27%) (P <0.001), resulting in a higher plaque burden for each CAC score compared with that in type 1 diabetic patients. CONCLUSIONS Although CAC scores and the prevalence of coronary atherosclerosis were similar between patients with type 1 and type 2 diabetes, CAD was more extensive in the latter. Also, a relatively higher proportion of noncalcified plaques was observed in patients with type 2 diabetes. These observations may be valuable in the development of targeted management strategies adapted to diabetes typ

Are coronary event rates declining slower in women than in men – evidence from two population-based myocardial infarction registers in Finland?

<p>Abstract</p> <p>Background</p> <p>Studies have suggested that the prevention and treatment of coronary heart disease may not have been as effective in women as in men. Therefore, we aimed to examine whether the incidence, attack rate and mortality of myocardial infarction (MI) events have declined less in women than in men.</p> <p>Methods</p> <p>Two large population-based MI registers, the FINAMI register and the Finnish Cardiovascular Disease Register (CVDR) were used for comparing the event rates among men and women aged ≥35 years in two time periods, 1994–1996 and 2000–2002.</p> <p>Results</p> <p>In the FINAMI register a total of 5,252 events were recorded in men and 4,898 in women. Corresponding numbers in the CVDR were 78,709 and 70,464. Both FINAMI and CVDR data suggested smaller declines in incidence and attack rate of MI events in women than in men. In CVDR data the decline in mortality was also smaller in women than in men, while in FINAMI data this difference did not reach statistical significance. In the large CVDR data set, negative binomial regression models revealed smaller declines in incidence (p = 0.006), attack rate (p = 0.008) and mortality (p = 0.04) in women than in men aged <55 years. In persons ≥55 years no difference was observed between women and men.</p> <p>Conclusion</p> <p>The incidence and attack rate of MI events have declined less in women aged <55 than in men of similar age. In older persons no significant differences were observed. Further studies are warranted to find out the reasons why the development has been less favourable for young women than for men.</p

The Genotype Specific Competitive Ability Does Not Correlate with Infection in Natural Daphnia magna Populations

Different evolutionary hypotheses predict a correlation between the fitness of a genotype in the absence of infection and the likelihood to become infected. The cost of resistance hypothesis predicts that resistant genotypes pay a cost of being resistant and are less fit in the absence of parasites. The inbreeding-infection hypothesis predicts that the susceptible individuals are less fit due to inbreeding depression.Here we tested if a host's natural infection status was associated with its fitness. First, we experimentally confirmed that cured but formerly infected Daphnia magna are genetically more susceptible to reinfections with Octosporea bayeri than naturally uninfected D. magna. We then collected from each of 22 populations both uninfected and infected D. magna genotypes. All were treated against parasites and kept in their asexual phase. We estimated their relative fitness in an experiment against a tester genotype and in another experiment in direct competition. Consistently, we found no difference in competitive abilities between uninfected and cured but formerly infected genotypes. This was the case both in the presence as well as in the absence of sympatric parasites during the competition trials.Our data do not support the inbreeding-infection hypothesis. They also do not support a cost of resistance, however ignoring other parasite strains or parasite species. We suggest as a possible explanation for our results that resistance genes might segregate largely independently of other fitness associated genes in this system

Acute myocardial infarction incidence and hospital mortality: routinely collected national data versus linkage of national registers

Background and Objective To compare levels of and trends in incidence and hospital mortality of first acute myocardial infarction (AMI) based on routinely collected hospital morbidity data and on linked registers. Cases taken from routine hospital data are a mix of patients with recurrent and first events, and double counting occurs when cases are admitted for an event several times during 1 year. By linkage of registers, recurrent events and double counts can be excluded. Study Design and Setting In 1995 and 2000, 28,733 and 25,864 admissions for AMI were registered in the Dutch national hospital discharge register. Linkage with the population register yielded 21,565 patients with a first AMI in 1995 and 20,414 in 2000. Results In 1995 and 2000, the incidence based on the hospital register was higher than based on the linked registers in men (22% and 23% higher) and women (18% and 20% higher). In both years, hospital mortality based on the hospital register and on linked registers was similar. The decline in incidence between 1995 and 2000 was comparable whether based on standard hospital register data or linked data (18% and 20% in men, 15% and 17% in women). Similarly, the decline in hospital mortality was comparable using either approach (11% and 9% in both men and women). Conclusion Although the incidence based on routine hospital data overestimates the actual incidence of first AMI based on linked registers, hospital mortality and trends in incidence and hospital mortality are not changed by excluding recurrent events and double counts. Since trends in incidence and hospital mortality of AMI are often based on national routinely collected data, it is reassuring that our results indicate that findings from such studies are indeed valid and not biased because of recurrent events and double counts