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

Novel renal markers for the assessment of renal integrity in patients undergoing knee arthroplasty - a pilot study

The feasibility of novel kidney injury biomarkers in consecutive patients having total knee arthroplasty with local infiltration analgesia was evaluated.\nWe enrolled 30 patients scheduled for elective unilateral total knee arthroplasty. Paired plasma and urine samples were taken before surgery and at 4 h, 24 h and 48 h after surgery to measure creatinine, cystatin C, neutrophil gelatinase associated lipocalin, kidney injury molecule-1, interleukin-18 and liver-type fatty acid-binding protein.\nAt baseline, 13 subjects had normal kidney function, 15 had mild and two had moderate kidney failure evaluated by calculated glomerular filtration rate. None of the subjects had all measured novel renal markers below proposed cut-off concentrations. Altogether 28/30 subjects had one (n = 3), two (n = 7) or three (n = 18) plasma neutrophil gelatinase associated lipocalin values above normal. In seven of these 28 subjects plasma creatinine, calculated glomerular filtration rate and plasma cystatin C were within the reference values. Five subjects had a low urine output, < 0.5 mL/h, indicating transient acute kidney injury, four of these had high plasma neutrophil gelatinase associated lipocalin and one high plasma cystatin C.\nIn the present study plasma neutrophil gelatinase associated lipocalin was elevated in most subjects with total knee arthroplasty and local infiltration analgesia as a marker of possible renal proximal tubular injury. Five subjects had transient low urine output, but none developed renal deterioration requiring treatment.\nBACKGROUND\nMETHODS\nRESULTS\nCONCLUSION

Willow-characterised shrub vegetation in tundra and its relation to abiotic, biotic and anthropogenic factors

Abstract Deciduous shrubs form the tallest type of vegetation in arctic-alpine areas and are important for ecosystem function. In the southern part of the Eurasian tundra zone, willows (Salix spp.) are the most common species in the shrub layer. In the alpine areas of Northern Fennoscandia, willow shrubs are characteristic to areas between tree line and treeless tundra heaths. Vertical structure and composition of willow-characterized tundra vegetation is affected by a variety of ecological factors including climate and herbivory. In turn, the abundance of the willow canopy affects understory species in several ways that still remain inadequately understood. In this PhD work I describe compositional differentiation of willow-characterized vegetation by using a large data set spanning from north-western Fennoscandia to the Yamal Peninsula in north-western Siberia. I studied environmental factors affecting willow-characterized vegetation and willow growth by using correlative analyses. The factors under investigation were latitude, distance from the sea, depth of thaw, position in the slope, industrial disturbance and reindeer grazing. In addition, I examined the relationships between the shrub biomass estimate and composition and species richness of understory vegetation. The effects of reindeer grazing on vegetation in an alpine forest-tundra ecotone were studied experimentally using reindeer-proof exclosures. I found that willow-characterized vegetation is floristically variable and comprises at least eight vegetation types. The most abundant willow thickets typically have a forb-rich understory. The growth of willow increased along with increasing summer temperatures. However the height of willow was more determined by distance from the sea, thaw depth and slope position. Reindeer grazing decreased the abundance of willow and changed the composition of understory vegetation. In addition, industrial activities were detected to have destructed shrub vegetation and turned it into graminoid-dominated vegetation. Shrub canopies facilitated forbs but decreased the cover of all the other groups including dwarf shrubs, bryophytes and lichens. The species richness of vegetation decreased along with increasing shrub abundance. My study shows that arctic-alpine willow vegetation is more diverse than previously thought. There is a predictable relationship between summer temperatures and willow growth. However, the results also show that there are many factors, both physical and anthropogenic, that are likely to complicate this pattern. Most important of these counteracting effects are industrial activities and reindeer grazing. In the areas where shrubs grow in abundance, the species richness of understory vegetation is likely to decrease and forbs are likely to replace other tundra species

Novel renal markers for the assessment of renal integrity in patients undergoing knee arthroplasty – a pilot study

Abstract Background The feasibility of novel kidney injury biomarkers in consecutive patients having total knee arthroplasty with local infiltration analgesia was evaluated. Methods We enrolled 30 patients scheduled for elective unilateral total knee arthroplasty. Paired plasma and urine samples were taken before surgery and at 4 h, 24 h and 48 h after surgery to measure creatinine, cystatin C, neutrophil gelatinase associated lipocalin, kidney injury molecule-1, interleukin-18 and liver-type fatty acid-binding protein. Results At baseline, 13 subjects had normal kidney function, 15 had mild and two had moderate kidney failure evaluated by calculated glomerular filtration rate. None of the subjects had all measured novel renal markers below proposed cut-off concentrations. Altogether 28/30 subjects had one (n = 3), two (n = 7) or three (n = 18) plasma neutrophil gelatinase associated lipocalin values above normal. In seven of these 28 subjects plasma creatinine, calculated glomerular filtration rate and plasma cystatin C were within the reference values. Five subjects had a low urine output, < 0.5 mL/h, indicating transient acute kidney injury, four of these had high plasma neutrophil gelatinase associated lipocalin and one high plasma cystatin C. Conclusions In the present study plasma neutrophil gelatinase associated lipocalin was elevated in most subjects with total knee arthroplasty and local infiltration analgesia as a marker of possible renal proximal tubular injury. Five subjects had transient low urine output, but none developed renal deterioration requiring treatment

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.Peer reviewe