Molecular characterization of anion exchangers in the cochlea

Anion exchange proteins (AE) in the inner ear have been the focus of attention for some time. They have been suggested to play a role as anion exchangers for the regulation of endolymphatic pH or as anion exchangers and anchor proteins for the maintenance of the shape and turgor of outer hair cells, and they also have been discussed as a candidate protein for motile hair cell responses that follow high-frequency stimulation. The existence of anion exchangers in hair cells and the specific isoforms which are expressed in hair cells and the organ of Corti is controversial. Using a polyclonal antibody to AE1 (AB1992, Chemicon), we immunoprecipitated a 100 kDa AE polypeptide in isolated outer hair cells which, due to its glycosylation, is comprised of AE2 than AE1 isoforms. We confirmed AE2 expression in outer hair cells with the help of subtype-specific monoclonal and polyclonal antibodies to AE, AE subtype-specific primers and AE subtype-specific cDNA and found glycosylated truncated as well as full-length AE2 isoforms. No AE1 or AE3 subtypes were noted in outer hair cells. In contrast, AE2 and AE3 but not AE1 subtypes were seen in supporting cells of the organ of Corti. Their expression preceded the development of cochlear function, coincident with the establishment of the endocochlear potential and the differentiation of supporting cells. While most developmental processes in the inner ear usually begin in the basal cochlear turn, the AE2 expression in outer hair cells (but not that of AE2 and AE3 in supporting cells) progressed from the apical to the basal cochlear turn, reminiscent of the maturation of frequency-dependency. Irrespective of their presumed individual role as either anion exchanger, anchor protein or motility protein, the differential expression and developmental profile of these proteins suggest a most important role of anion exchange proteins in the development of normal hearing. These findings may also provide novel insights into AE function in general.     

Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations

Antibodies are valuable tools in the laboratory and clinic. Antibodies include those secreted by a single clone of B lymphocytes, termed monoclonal antibodies, and those produced by a mixture of various B lymphocyte clones, termed polyclonal antibodies. Both products have become essential instruments in fundamental immunological research, immunohistochemistry, diagnostic testing, and vaccine quality control. Antibody production requires a substantial number of animals, and the animals are subjected to a number of invasive procedures such as antigen injection and blood collection. However, by carefully designing an immunization protocol and by optimizing the immunization response, it is possible to minimize animals' pain and distress while obtaining optimal immune responses. In this article, the critical steps in the production of polyclonal and monoclonal antibodies are described, specifically including selection of the animal species and its age, injection protocol, and ascites tapping. Recommendations are provided for optimizing the immunization response.     

Some aspects of induction and termination of diapause in a Greek strain of the miteTetranychus cinnabarinus (Boisduval) Boudreaux, 1956 (Acari: Tetranychidae)

A Greek strain of the miteTetranychus cinnabarinus, collected from ivy (Hedera spec.) in Thessaloniki (41 °N), exhibits a facultative, imaginal diapause. Diapause is induced by photoperiod and the photoperiodic response is of the long-day type. The critical daylength is 12.5 h at 19 °C. A period of chilling is not necessary for the termination of diapause under long-day conditions. Diapausing females are sensitive to photoperiod at least during the first 11/2 month of diapause.     

The Bizarre Inflorescence of Norantea brasiliensis (Marcgraviaceae): Visits of Hovering and Perching Birds1

The pollination biology of Norantea brasiliensis (Marcgraviaceae) was studied in the rain forest of southeastern Brazil. This plant presents bizarre, brush-type racemous inflorescences bearing numerous flowers and extrafloral cup-shaped nectaries. Flower anthesis is diurnal, nectar production is continuous and copious, and the sticky pollen is readily removed by visitors during the first morning hours. The ruby-coloured inflorescences were visited by eight species of hummingbirds (Trochilidae), and 10 species of passerine birds (three Coerebidae and seven Thraupidae). Hummingbirds hovered while probing for nectar and touched flowers occasionally, whereas passerine birds perched and made contact with flowers habitually. Due to differences in flower-visiting and general foraging behaviour, perching birds act as better pollen vectors than hovering birds. The inflorescence of Norantea brasiliensis seems well fitted for pollination by passerine birds, and the hexose-dominated nectar supports this idea. Pollination syndrome trends within Marcgraviaceae may stem from insect-pollinated, condensed and spike-like inflorescences which would give rise to bird-pollinated, brush-type inflorescences. From the same basic condensed inflorescence, bat-pollinated umbelliform inflorescence may be derived from bird-pollinated, pendulous and corymb-like inflorescences. These postulated inflorescence types are found among the extant species of Marcgraviaceae.     

A Cluster-Randomized Trial of Two Strategies to Improve Antibiotic Use for Patients with a Complicated Urinary Tract Infection

Background

Up to 50% of hospital antibiotic use is inappropriate and therefore improvement strategies are urgently needed. We compared the effectiveness of two strategies to improve the quality of antibiotic use in patients with a complicated urinary tract infection (UTI).

Methods

In a multicentre, cluster-randomized trial 19 Dutch hospitals (departments Internal Medicine and Urology) were allocated to either a multi-faceted strategy including feedback, educational sessions, reminders and additional/optional improvement actions, or a competitive feedback strategy, i.e. providing professionals with non-anonymous comparative feedback on the department’s appropriateness of antibiotic use. Retrospective baseline- and post-intervention measurements were performed in 2009 and 2012 in 50 patients per department, resulting in 1,964 and 2,027 patients respectively. Principal outcome measures were nine validated guideline-based quality indicators (QIs) that define appropriate antibiotic use in patients with a complicated UTI, and a QI sumscore that summarizes for each patient the appropriateness of antibiotic use.

Results

Performance scores on several individual QIs showed improvement from baseline to post-intervention measurements, but no significant differences were found between both strategies. The mean patient’s QI sum score improved significantly in both strategy groups (multi-faceted: 61.7% to 65.0%, P = 0.04 and competitive feedback: 62.8% to 66.7%, P = 0.01). Compliance with the strategies was suboptimal, but better compliance was associated with more improvement.

Conclusion

The effectiveness of both strategies was comparable and better compliance with the strategies was associated with more improvement. To increase effectiveness, improvement activities should be rigorously applied, preferably by a locally initiated multidisciplinary team.

Trial Registration

Nederlands Trial Register 1742     

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as ‘good neighbors’ in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of ‘good neighbors’ in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.     

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.