Structural comparison of the 68 kDa laminin-binding protein and 5'-nucleotidase from chicken muscular sources: evidence against a gross structural similarity of both proteins

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5'-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5'-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5'-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may posses a few common peptides of yet unknown sequence and length.     

p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo

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Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes

Bloom''s syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM–DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.     

Quality Indicators for Safe Medication Preparation and Administration: A Systematic Review

Background

One-third of all medication errors causing harm to hospitalized patients occur in the medication preparation and administration phase, which is predominantly a nursing activity. To monitor, evaluate and improve the quality and safety of this process, evidence-based quality indicators can be used.

Objectives

The aim of study was to identify evidence-based quality indicators (structure, process and outcome) for safe in-hospital medication preparation and administration.

Methods

MEDLINE, EMBASE and CINAHL were searched for relevant studies published up to January 2015. Additionally, nine databases were searched to identify relevant grey literature. Two reviewers independently selected studies if (1) the method for quality indicator development combined a literature search with expert panel opinion, (2) the study contained quality indicators on medication safety, and (3) any of the quality indicators were applicable to hospital medication preparation and administration. A multidisciplinary team appraised the studies independently using the AIRE instrument, which contains four domains and 20 items. Quality indicators applicable to in-hospital medication preparation and administration were extracted using a structured form.

Results

The search identified 1683 studies, of which 64 were reviewed in detail and five met the inclusion criteria. Overall, according to the AIRE domains, all studies were clear on purpose; most of them applied stakeholder involvement and used evidence reasonably; usage of the indicator in practice was scarcely described. A total of 21 quality indicators were identified: 5 structure indicators (e.g. safety management and high alert medication), 11 process indicators (e.g. verification and protocols) and 5 outcome indicators (e.g. harm and death). These quality indicators partially cover the 7 rights.

Conclusion

Despite the relatively small number of included studies, the identified quality indicators can serve as an excellent starting point for further development of nursing specific quality indicators for medication safety. Especially on the right patient, right route, right time and right documentation there is room future development of quality indicators.     

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8 +/- 1.2 microM) was significantly lower than its cytostatic activity; CC50 (261.3 +/- 14.5 microM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.     

Age Related Differences in Dynamics of Specific Memory B Cell Populations after Clinical Pertussis Infection

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (B_mem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and B_mem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of B_mem cell populations; higher levels of B_mem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects'' levels of specific B_mem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific B_mem cell and plasma IgG levels.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Contribution of the Ly49E Natural Killer Receptor in the Immune Response to Plasmodium berghei Infection and Control of Hepatic Parasite Development

Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial hepatic stage of infection by production of the T_H1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8⁺ T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6–7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5⁻ liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.     

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody? ALX-0061 supports its clinical development in rheumatoid arthritis

IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.     

On the Subjective Acceptance during Cardiovascular Magnetic Resonance Imaging at 7.0 Tesla

Purpose

This study examines the subjective acceptance during UHF-CMR in a cohort of healthy volunteers who underwent a cardiac MR examination at 7.0T.

Methods

Within a period of two-and-a-half years (January 2012 to June 2014) a total of 165 healthy volunteers (41 female, 124 male) without any known history of cardiac disease underwent UHF-CMR. For the assessment of the subjective acceptance a questionnaire was used to examine the participants experience prior, during and after the UHF-CMR examination. For this purpose, subjects were asked to respond to the questionnaire in an exit interview held immediately after the completion of the UHF-CMR examination under supervision of a study nurse to ensure accurate understanding of the questions. All questions were answered with “yes” or “no” including space for additional comments.

Results

Transient muscular contraction was documented in 12.7% of the questionnaires. Muscular contraction was reported to occur only during periods of scanning with the magnetic field gradients being rapidly switched. Dizziness during the study was reported by 12.7% of the subjects. Taste of metal was reported by 10.1% of the study population. Light flashes were reported by 3.6% of the entire cohort. 13% of the subjects reported side effects/observations which were not explicitly listed in the questionnaire but covered by the question about other side effects. No severe side effects as vomiting or syncope after scanning occurred. No increase in heart rate was observed during the UHF-CMR exam versus the baseline clinical examination.

Conclusions

This study adds to the literature by detailing the subjective acceptance of cardiovascular magnetic resonance imaging examinations at a magnetic field strength of 7.0T. Cardiac MR examinations at 7.0T are well tolerated by healthy subjects. Broader observational and multi-center studies including patient cohorts with cardiac diseases are required to gain further insights into the subjective acceptance of UHF-CMR examinations.