Large-scale preparation of γ-crystallin and fractionation by chromatofocusing

Large quantities of calf γ-crystallin can be prepared by a single and rapid salting-out procedure. The final product is indistinguishable from the γ-crystallin fraction obtained after gel filtration of a 15000 g lens protein supernatant over Sephadex G 200. Further fractionation is achieved by the mild procedure of chromatofocusing yielding six to eight subfractions. The latter have been characterized by polyacrylamide gel electrophoresis (PAGE) in 6 m-urea, SDS-gel electrophoresis, partial digestion with Staphylococcus aureus protease and amino acid analysis.     

Quantitative T*2‐mapping based on multi‐slice multiple gradient echo flash imaging: Retrospective correction for subject motion effects

Numerous clinical and research applications for quantitative mapping of the effective transverse relaxation time T*₂ have been described. Subject motion can severely deteriorate the quality and accuracy of results. A correction method for T*₂ maps acquired with multi‐slice multiple gradient echo FLASH imaging is presented, based on acquisition repetition with reduced spatial resolution (and consequently reduced acquisition time) and weighted averaging of both data sets, choosing weighting factors individually for each k‐space line to reduce the influence of motion. In detail, the procedure is based on the fact that motion artifacts reduce the correlation between acquired and exponentially fitted data. A target data set is constructed in image space, choosing the data yielding best correlation from the two acquired data sets. The k‐space representation of the target is subsequently approximated as linear combination of original raw data, yielding the required weighting factors. As this method only requires a single acquisition repetition with reduced spatial resolution, it can be employed on any clinical system offering a suitable sequence with export of modulus and phase images. Experimental results show that the method works well for sparse motion, but fails for strong motion affecting the same k‐space lines in both acquisitions. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Delayed formation of actin filaments in the outer pillar head plate of VASP-/- mice

Pillar cells with their rich network of tubulin and actin filaments have been reported to contribute to the rigidity of the organ of Corti. As the earliest expression of the actin filament enhancer vasodilator-stimulated phosphoprotein (VASP) in the outer pillar head plate has been found to be associated with the onset of hearing, we tested hearing development in VASP-/- compared to wild-type mice. Performing measurements of auditory brainstem responses on postnatal days (P) P14 and P21, we detected statistically significantly higher thresholds in VASP-/- compared to wild-type mice at P14, but no hearing differences at P21. Staining for prestin and synaptophysin at P12 in morphologically regularly developed cochleae of VASP-/- mice provided an immature prestin protein pattern but no evidence of developmental delay in hair cell innervations. Regularly intense staining of actin filaments in the outer pillar head plate was found only in wild-type but not in VASP-/- mice at P14. At P21, intensive actin filament staining was also observed in the outer pillar head plates of VASP-/- mice. The delayed hearing development in VASP-/- mice is supposed to be caused by a delayed formation of actin filaments in the outer pillar head plate indicating the importance of appropriate pillar cell stiffness in cochlear mechanics.     

Risk factors and impact of major bleeding in critically ill patients receiving heparin thromboprophylaxis

Purpose

Bleeding frequently complicates critical illness and may have serious consequences. Our objectives are to describe the predictors of major bleeding and the association between bleeding and mortality in medical–surgical critically ill patients receiving heparin thromboprophylaxis.

Methods

We prospectively studied patients from 67 intensive care units and six countries enrolled in a thromboprophylaxis trial (NCT00182143) comparing dalteparin with unfractionated heparin. Patients with trauma, orthopedic surgery or neurosurgery were excluded. Trained research coordinators used a validated tool to document bleeding, which underwent duplicate independent blinded adjudication. Major bleeding was defined as hypovolemic shock, bleeding into critical sites, requiring an invasive intervention or transfusion of at least two units of red blood cells, or associated with hypotension or tachycardia in the absence of other causes. Adjusted Cox proportional hazard regression analysis was used to identify major bleeding predictors and the association between bleeding and mortality.

Results

Among 3,746 patients, bleeding occurred in 208 [5.6 %, 95 % confidence interval (CI) 4.9–6.3 %]. Time-dependent predictors were prolonged activated partial thromboplastin time [hazard ratio (HR) 1.10, 1.05–1.14 per 10 s increase], lower platelet count (HR 1.16, 1.09–1.24 per 50 × 10⁹/L decrease), therapeutic heparin (HR 3.26, 1.72–6.17), antiplatelet agents (HR 1.38, 1.02–1.88), renal replacement therapy (HR 1.75, 1.20–2.56), and recent surgery (HR 1.64, 1.01–2.65). Type of pharmacologic thromboprophylaxis was not associated with bleeding. Patients with bleeding had a higher risk of in-hospital death (HR 2.09, 1.69–2.57).

Conclusions

As major bleeding has modifiable risk factors and is associated with in-hospital mortality, strategies to mitigate these factors should be evaluated in critically ill patients.     

Rab8b GTPase, a protein transport regulator, is an interacting partner of otoferlin, defective in a human autosomal recessive deafness form

Mutations within OTOF encoding otoferlin lead to a recessive disorder called DFNB9. Several studies have indicated otoferlin's association with ribbon synapses of cochlear sensory hair cells, as well as data showing the protein's presence in neurons, nerve fibers and hair cells, suggesting a more ubiquitous function. Otoferlin's co-localization not only with ribbon synaptic proteins, but also with additional endosomal (EEA1) or Golgi proteins (GM130) were motivation for a search for further binding partners of otoferlin by a yeast two-hybrid screen in a rodent cochlear cDNA library (P3-P15). This screen identified Rab8b GTPase as a novel interacting partner, substantiated by transient co-expression and co-localization in HEK 293 cells and co-immunoprecipitation of the complex using tagged proteins in vitro and native proteins from cochlea. This finding implies that otoferlin could be a part of components contributing to trans-Golgi trafficking.     

Renal interstitial fibrosis (RIF): II. Ultrasound follow up study of single uranyl nitrate administration causing renal dysfunction in rats – comparison with histologic and functional renal parameters

A single administration of uranyl nitrate (UN; 0.5 mg/100 g b. wt. i.p.) to adult female Wistar rats reliably induces renal interstitial fibrosis (RIF) providing an experimental model to investigate therapeutic strategies. It was the aim of this study to further characterise a rat model of UN induced RIF which we have studied previously (APPENROTH et al. 2001) by the comparison of functional parameters with ultrasonographic examination over a period of 30 weeks after injury. In the acute phase after UN administration (between days 2 and 17) signs of inflammation (increase in renal blood flow, swelling of renal cortex, enlargement of renal pelvis) could be detected by ultrasound. After four weeks UN led to functional changes (decreased creatinine clearance, increased urinary protein excretion and increased OH-proline concentration in renal tissue). In vitro, the accumulation of p-aminohippurate and the gluconeogenesis were reduced. In accordance with the functional changes, distinct ultrasonographic abnormalities could be seen between weeks 10 and 30 after UN with regard to changes in kidney size and shape, reduced renal perfusion and enlargement of renal pelvis. The sensitivity of ultrasonography in small laboratory animals is limited and most useful for follow-up studies of acute renal changes after administration of nephrotoxins. Ultrasonography can not be recommended for non-invasive screening of the progression of chronic renal failure.     

Enhanced memory B-cell immune responses after a second acellular pertussis booster vaccination in children 9 years of age

Whooping cough has made its comeback and the incidence of pertussis in countries with widespread pertussis vaccination is most prominent in individuals above 9 years of age. To control the burden of infection, several countries already introduced acellular pertussis (aP) booster vaccination in adolescents and/or adults. However, antibody levels wane rapidly after vaccination even at older age. In this longitudinal study we investigated the effect of a second aP booster on the pertussis-specific memory B-cell immunity in children 9 years of age that have previously been vaccinated according to the national immunization program. Longitudinal blood samples were taken before, one month and one year after the booster. Purified B-cells were polyclonally stimulated and frequencies of memory B-cells were identified by ELISPOT-assays specific for various pertussis antigens. In addition, IgG levels and avidity indices were measured with fluorescent bead-based multiplex immunoassays.Starting with low pertussis-specific antibody and memory B-cell levels, a typical booster response was measured at one month after vaccination with increased antibody and memory B-cell responses. Although these responses declined slightly after one year, they substantially exceeded pre-booster levels and the avidity indices of the anti-pertussis antibodies remained high. Furthermore, high numbers of pertussis-specific memory B-cells at one-month post-booster correlate quite reliably with the corresponding high antibody response at one-year follow-up. In conclusion, booster vaccination in children 9 years of age induced an enhanced pertussis-specific memory immune response that sustained at least for one year. Therefore, this study supports the introduction of booster vaccination in older age groups.     

Information on vaccination: meeting the needs of unvaccinated youngsters in the Netherlands

To improve vaccination coverage in the Netherlands, compulsory consultation of the youth health service has been suggested for unvaccinated youngsters. It is assumed that sound medical arguments will convince them to accept vaccination. We assessed the need for information of the highest risk group, the unvaccinated orthodox protestant youngsters. Only 21% of over 600 respondents were interested in medical aspects of vaccination, whereas >50% were interested in religious aspects. Their preferred information source was a Christian organization, not the youth health service. Our study shows the importance of exploration of the target group before introducing a new policy.     

Temporale Enzephalozelen – eine neue Ursache von Temporallappenepilepsien?

Clinical Epileptology - Temporale Enzephalozelen sind bisher nur in wenigen Fällen als Ursache von symptomatischen Epilepsien beschrieben worden. Zunehmende Beschreibungen in Fallserien weisen...