Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

Abstract The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.     

Molecular Phylogeny of Some European Heteronemertean (Nemertea) Species and the Monophyletic Status ofRiseriellus, Lineus,andMicrura

The 16S rRNA mitochondrial gene was used to reconstruct the relationships among 10 heteronemertean species (subclass Heteronemertea, phylum Nemertea);Lineus ruberandL. viridisare represented by more than one specimen to assess intraspecific variation in these enigmatic species, and the analysis includes in total 14 terminal taxa incorporating one palaeonemertean species (Tubulanus annulatus) for outgroup rooting. The aligned sequences were subjected to maximum parsimony, maximum-likelihood, and neighbor-joining analyses to estimate the phylogenetic relationship of the species. The results were concordant from all analyses and indicate that neitherLineusnorMicruraare monophyletic taxa, and that there is no support from a phylogenetic point of view to establish the monotypic genusRiseriellus.     

Effect of grazing by a neotropical copepod, Notodiaptomus, on a natural cyanobacterial assemblage and on toxic and non-toxic cyanobacterial strains

The neotropical, freshwater copepod Notodiaptomus iheringi decreasedthe growth of small colonies in cyanobacteria-dominated plankton,indicating that they can be efficiently ingested and that theymay represent an important food source for this copepod in reservoirsdominated by cyanobacteria. Cultured, colonial, toxic Microcystisaeruginosa was not affected, indicating that toxicity may causea decrease in its ingestion by N. iheringi.     

Nitrogen mineralization and denitrification as influenced by crop residue particle size

Managing the crop residue particle size has the potential to affect N conservation in agricultural systems. We investigated the influence of barley (Hordeum vulgare) and pea (Pisum sativum) crop residue particle size on N mineralization and denitrification in two laboratory experiments. Experiment 1: ¹⁵N-labelled ground (3 mm) and cut (25 mm) barley residue, and microcrystalline cellulose+glucose were mixed into a sandy loam soil with additional inorganic N. Experiment 2: inorganic¹⁵ N and C₂H₂ were added to soils with barley and pea material after 3, 26, and 109 days for measuring gross N mineralization and denitrification.Net N immobilization over 60 days in Experiment 1 cumulated to 63 mg N kg^-1 soil (ground barley), 42 (cut barley), and 122 (cellulose+glucose). More N was seemingly net mineralized from ground barley (3.3 mg N kg^-1 soil) than from cut barley (2.7 mg N kg^-1 soil). Microbial biomass peaked at day 4 with the barley treatments and at day 14 with the cellulose+glucose whereafter the biomass leveled out at values 79 mg C kg^-1 (ground), 104 (cut), and 242 (cellulose+glucose) higher than for the control soil. Microbial growth yields were similar for the two barley treatments, ca. 60 mg C g^-1 substrate C added, which was lower than the 142 mg C g^-1 C added with cellulose+glucose. This suggests that the 75% (w/w) holocelluloses and sugars contained with the barley material remained physically protected despite grinding. In Experiment 2 gross mineralization on day 3 was 4.8 mg N kg^-1 d^-1 with ground pea, twice as much as for all other treatments. On day 26 the treatment with ground barley had the greatest gross N mineralization. In static cores ground barley denitrified 11-fold more than did cut barley, whereas denitrification was similar for the two pea treatments. In suspensions denitrification was similar for the two treatments both with barley and pea residue.We conclude that the higher microbial activity associated with the initial decomposition of ground plant material is due to a more intimate plant residue-soil contact. On the long term, grinding the plant residues has no significant effect on N dynamics.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Time‐dependent climate impact and energy efficiency of combined heat and power production from short‐rotation coppice willow using pyrolysis or direct combustion

A life cycle assessment of a Swedish short‐rotation coppice willow bioenergy system generating electricity and heat was performed to investigate how the energy efficiency and time‐dependent climate impact were affected when the feedstock was converted into bio‐oil and char before generating electricity and heat, compared with being combusted directly. The study also investigated how the climate impact was affected when part of the char was applied to soil as biochar to act as a carbon sequestration agent and potential soil improver. The energy efficiencies were calculated separately for electricity and heat as the energy ratios between the amount of energy service delivered by the system compared to the amount of external energy inputs used in each scenario after having allocated the primary energy related to the inputs between the two energy services. The energy in the feedstock was not included in the external energy inputs. Direct combustion had the highest energy efficiency. It had energy ratios of 10 and 36 for electricity and heat, respectively. The least energy‐efficient scenario was the pyrolysis scenario where biochar was applied to soils. It had energy ratios of 4 and 12 for electricity and heat, respectively. The results showed that pyrolysis with carbon sequestration might be an option to counteract the current trend in global warming. The pyrolysis system with soil application of the biochar removed the largest amount of from the atmosphere. However, compared with the direct combustion scenario, the climate change mitigation potential depended on the energy system to which the bioenergy system delivered its energy services. A system expansion showed that direct combustion had the highest climate change mitigation potential when coal or natural gas were used as external energy sources to compensate for the lower energy efficiency of the pyrolysis scenario.     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Impact of electro-acupuncture and physical exercise on hyperandrogenism and oligo/amenorrhea in women with polycystic ovary syndrome: a randomized controlled trial

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism, oligo/amenorrhea, and polycystic ovaries. We aimed to determine whether low-frequency electro-acupuncture (EA) would decrease hyperandrogenism and improve oligo/amenorrhea more effectively than physical exercise or no intervention. We randomized 84 women with PCOS, aged 18-37 yr, to 16 wk of low-frequency EA, physical exercise, or no intervention. The primary outcome measure changes in the concentration of total testosterone (T) at week 16 determined by gas and liquid chromatography-mass spectrometry was analyzed by intention to treat. Secondary outcome measures were changes in menstrual frequency; concentrations of androgens, estrogens, androgen precursors, and glucuronidated androgen metabolites; and acne and hirsutism. Outcomes were assessed at baseline, after 16 wk of intervention, and after a 16-wk follow-up. After 16 wk of intervention, circulating T decreased by -25%, androsterone glucuronide by -30%, and androstane-3α,17β-diol-3-glucuronide by -28% in the EA group (P = 0.038, 0.030, and 0.047, respectively vs. exercise); menstrual frequency increased to 0.69/month from 0.28 at baseline in the EA group (P = 0.018 vs. exercise). After the 16-wk follow-up, the acne score decreased by -32% in the EA group (P = 0.006 vs. exercise). Both EA and exercise improved menstrual frequency and decreased the levels of several sex steroids at week 16 and at the 16-wk follow-up compared with no intervention. Low-frequency EA and physical exercise improved hyperandrogenism and menstrual frequency more effectively than no intervention in women with PCOS. Low-frequency EA was superior to physical exercise and may be useful for treating hyperandrogenism and oligo/amenorrhea.