Genetic Diversity of the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Drug Users in Amsterdam: Are They Still at Risk for HIV?

Background and Aims

To examine whether drug users (DU) in the Amsterdam Cohort Study (ACS) are still at risk for HIV, we studied trends in HIV incidence and injecting and sexual risk behaviour from 1986 to 2011.

Methods

The ACS is an open, prospective cohort study on HIV. Calendar time trends in HIV incidence were modelled using Poisson regression. Trends in risk behaviour were modelled via generalized estimating equations. In 2010, a screening for STI (chlamydia, gonorrhoea and syphilis) was performed. Determinants of unprotected sex were studied using logistic regression analysis.

Results

The HIV incidence among 1298 participants of the ACS with a total follow-up of 12,921 person-years (PY) declined from 6.0/100 PY (95% confidence interval [CI] 3.2–11.1) in 1986 to less than 1/100 PY from 1997 onwards. Both injection and sexual risk behaviour declined significantly over time. Out of 197 participants screened for STI in 2010–2011, median age 49 years (IQR 43–59), only 5 (2.5%) were diagnosed with an STI. In multivariable analysis, having a steady partner (aOR 4.1, 95% CI 1.6–10.5) was associated with unprotected sex. HIV-infected participants were less likely to report unprotected sex (aOR 0.07, 95% CI 0.02–0.37).

Conclusions

HIV incidence and injection risk behaviour declined from 1986 onwards. STI prevalence is low; unprotected sex is associated with steady partners and is less common among HIV-infected participants. These findings indicate a low transmission risk of HIV and STI, which suggests that DU do not play a significant role in the current spread of HIV in Amsterdam.     

Flavonols are not essential for fertilization in Arabidopsis thaliana

Flavonols are plant metabolites suggested to serve a vital role in fertilization of higher plants. Petunia and maize plants mutated in their flavonol biosynthesis are not able to set seed after self-pollination. We have investigated the role of these compounds in Arabidopsis thaliana. Like in all other plant species, high levels of flavonols could be detected in pollen of wild-type A. thaliana. No flavonols were detected in reproductive organs of the A. thaliana tt4 mutant in which the chs gene is mutated. Surprisingly, this mutant did set seed after self-fertilization and no pollen tube growth aberrations were observed in vivo. The role of flavonols during fertilization of Arabidopsis is discussed.Abbreviations CHS chalcone synthase - TLC thin-layer chromatography     

Preperoxisomal vesicles can form in the absence of Pex3

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.     

Macrophyte-related shifts in the nitrogen and phosphorus contents of the different trophic levels in a biomanipulated shallow lake

Lake Zwemlust, a small highly eutrophic lake, was biomanipulated without reducing the external nutrient loading, and the effects were studied for four years. In this paper we pay special attention to the shifts in relative distribution of nitrogen and phosphorus in the different trophic levels and to the changes in growth limitation of the autotrophs.Despite of the high external nutrient loads to the lake (ca 2.4 g P m^–2 y^–1 and 9.6 g N m^–2 y^–1), the effects of biomanipulation on the lake ecosystem were pronounced. Before biomanipulation no submerged vegetation was present in the lake and P and N were stored in the phytoplankton (44% N, 47% P), fish (33% N, 9% P) and in dissolved forms (23% N, 44% P). P and N contents in sediments were not determined. In the spring and summer following the biomanipulation (1987), zooplankton grazing controlled the phytoplankton biomass and about 90% of N and P were present in dissolved form in the water. From 1988 onwards submerged macrophyte stands continue to thrive, reducing the ammonium and nitrate concentrations in the water below detection levels. In July 1989 storage of N and P in the macrophytes reached 86% and 80%, respectively. Elodea nuttallii (Planchon) St.John, the dominant species in 1988 and 1989, acted as sink both for N and P during spring and early summer, withdrawing up to ca 60% of its N and P content from the sediment. At the end of the year only part of the N and P from the decayed macrophytes (ca 30% of N and 60% of P) was recovered in the water phase of the ecosystem (chiefly in dissolved forms). The rest remained in the sediment, although some N may have been released from the lake by denitrification.In summer 1990 only 30% of the N and P was found in the macrophytes (dominant species Ceratophyllum demersum L.), while ca 30% of N and P was again stored in phytoplankton and fish.     

Development and Validation of a Prediction Model for Tube Feeding Dependence after Curative (Chemo-) Radiation in Head and Neck Cancer

Background

Curative radiotherapy or chemoradiation for head and neck cancer (HNC) may result in severe acute and late side effects, including tube feeding dependence.The purpose of this prospective cohort study was to develop a prediction model for tube feeding dependence 6 months (TUBE_M6) after curative (chemo-) radiotherapy in HNC patients.

Patients and Methods

Tube feeding dependence was scored prospectively. To develop the multivariable model, a group LASSO analysis was carried out, with TUBE_M6 as the primary endpoint (n = 427). The model was then validated in a test cohort (n = 183). The training cohort was divided into three groups based on the risk of TUBE_M6 to test whether the model could be extrapolated to later time points (12, 18 and 24 months).

Results

Most important predictors for TUBE_M6 were weight loss prior to treatment, advanced T-stage, positive N-stage, bilateral neck irradiation, accelerated radiotherapy and chemoradiation. Model performance was good, with an Area under the Curve of 0.86 in the training cohort and 0.82 in the test cohort. The TUBE_M6-based risk groups were significantly associated with tube feeding dependence at later time points (p<0.001).

Conclusion

We established an externally validated predictive model for tube feeding dependence after curative radiotherapy or chemoradiation, which can be used to predict TUBE_M6.     

Ecophysiological response of Crambe maritima to airborne and soil-borne salinity

Background and Aims

There is a need to evaluate the salt tolerance of plant species that can be cultivated as crops under saline conditions. Crambe maritima is a coastal plant, usually occurring on the driftline, with potential use as a vegetable crop. The aim of this experiment was to determine the growth response of Crambe maritima to various levels of airborne and soil-borne salinity and the ecophysiological mechanisms underlying these responses.

Methods

In the greenhouse, plants were exposed to salt spray (400 mm NaCl) as well as to various levels of root-zone salinity (RZS) of 0, 50, 100, 200 and 300 mm NaCl during 40 d. The salt tolerance of Crambe maritima was assessed by the relative growth rate (RGR) and its components. To study possible salinity effects on the tissue and cellular level, the leaf succulence, tissue Na⁺ concentrations, Na⁺ : K⁺ ratio, net K⁺/Na⁺ selectivity, N, P, K⁺, Ca²⁺, Mg²⁺, proline, soluble sugar concentrations, osmotic potential, total phenolics and antioxidant capacity were measured.

Key Results

Salt spray did not affect the RGR of Crambe maritima. However, leaf thickness and leaf succulence increased with salt spray. Root zone salinities up to 100 mm NaCl did not affect growth. However, at 200 mm NaCl RZS the RGR was reduced by 41 % compared with the control and by 56 % at 300 mm NaCl RZS. The reduced RGR with increasing RZS was largely due to the reduced specific leaf area, which was caused by increased leaf succulence as well as by increased leaf dry matter content. No changes in unit leaf rate were observed but increased RZS resulted in increased Na⁺ and proline concentrations, reduced K⁺, Ca²⁺ and Mg²⁺ concentrations, lower osmotic potential and increased antioxidant capacity. Proline concentrations of the leaves correlated strongly (r = 0·95) with RZS concentrations and not with plant growth.

Conclusions

Based on its growth response, Crambe maritima can be classified as a salt spray tolerant plant that is sensitive to root zone salinities exceeding 100 mm NaCl.     

Finding the Needles in the Metagenome Haystack

In the collective genomes (the metagenome) of the microorganisms inhabiting the Earth’s diverse environments is written the history of life on this planet. New molecular tools developed and used for the past 15 years by microbial ecologists are facilitating the extraction, cloning, screening, and sequencing of these genomes. This approach allows microbial ecologists to access and study the full range of microbial diversity, regardless of our ability to culture organisms, and provides an unprecedented access to the breadth of natural products that these genomes encode. However, there is no way that the mere collection of sequences, no matter how expansive, can provide full coverage of the complex world of microbial metagenomes within the foreseeable future. Furthermore, although it is possible to fish out highly informative and useful genes from the sea of gene diversity in the environment, this can be a highly tedious and inefficient procedure. Microbial ecologists must be clever in their pursuit of ecologically relevant, valuable, and niche-defining genomic information within the vast haystack of microbial diversity. In this report, we seek to describe advances and prospects that will help microbial ecologists glean more knowledge from investigations into metagenomes. These include technological advances in sequencing and cloning methodologies, as well as improvements in annotation and comparative sequence analysis. More significant, however, will be ways to focus in on various subsets of the metagenome that may be of particular relevance, either by limiting the target community under study or improving the focus or speed of screening procedures. Lastly, given the cost and infrastructure necessary for large metagenome projects, and the almost inexhaustible amount of data they can produce, trends toward broader use of metagenome data across the research community coupled with the needed investment in bioinformatics infrastructure devoted to metagenomics will no doubt further increase the value of metagenomic studies in various environments.