Electrophysiological effects of aprindine on isolated heart preparations

The electrophysiological effects of aprindine, a new anti-arrhythmic drug, were investigated in isolated cardiac tissues of different animals. Diastolic depolarization and spontaneous firing were attenuated or abolished by aprindine. The action potential duration and effective refractory period were markedly shortened in Purkinje fibres. At the same concentrations, the action potential duration in atrial and ventricular muscle was not significantly altered. Membrane responsiveness curve was shifted to a more negative membrane potential. At concentrations above 5 mg/l, Purkinje fibres became inexcitable. Uptake and release of ¹⁴C-aprindine was studied in isolated heart preparations. Equilibration was not reached after an incubation period of 60 min and a final concentration of 10 times the concentration in the uptake fluid was attained after 60 min. The long-lasting effects on transmembrane potentials could be explained by the slow release of ¹⁴C-aprindine. A large fraction of the ¹⁴C-aprindine content was released with a time constant of 3 hr.The mechanism responsible for the in vivo anti-arrhythmic action and side effects are discussed.     

Hot stage extrusion of p-amino salicylic acid with EC using CO2 as a temporary plasticizer

The aim of the current research project was to explore the possibilities of combining pressurized carbon dioxide with hot stage extrusion during manufacturing of solid dispersions of the thermally labile p-aminosalicylic acid (p-ASA) and ethylcellulose 20cps (EC 20cps) and to evaluate the ability of the pressurized gas to act as a temporary plasticizer. The thermal stability of the p-ASA was investigated using DSC, TGA and HPLC. The compound decomposes completely upon melting. Below 110 degrees C and under atmospheric conditions, the compound is thermally stabile for 10min. Pressurized carbon dioxide was injected into a Leistritz Micro 18 intermeshing co-rotating twin-screw melt extruder using an ISCO 260D syringe pump. Carbon dioxide acted as plasticizer for p-ASA/EC 20cps, reducing the processing temperature during the hot stage extrusion process. HPLC showed that without carbon dioxide injection, approximately 17% of p-ASA degraded, while less than 5% degraded with CO(2) injection. The experiments clearly showed that injecting pressurized carbon dioxide broadens the application of hot stage extrusion to thermally labile compounds in a one step process.     

A new lupus anticoagulant neutralization test based on platelet-derived vesicles

We here present an easily standardizable and reproducible procedure which clearly separates lupus anticoagulants (LA) from coagulation factor inhibitors. This new LA neutralization test makes use of platelet-derived microvesicles which were prepared as follows: gel-filtered platelets (4 x 10(5)/microliters) were incubated with 60 microM of the calcium ionophore A23187 for 20 min at 37 degrees C. The vesicles were separated from the platelet aggregates by centrifugation at 1000 g for 10 min. The vesicle containing supernatant was then spun down at 15,000 g for 15 min, lyophilized and stored at -20 degrees C until used. The vesicles were resuspended in plasma from normal individuals, from patients with LA activity, from patients with factor VIII inhibitors, from patients with congenital factor deficiencies and from patients receiving oral anticoagulants or intravenous heparin. A kaolin clotting time was performed in the absence (KCT) or presence of these vesicles (KCTves) and the ratios of these times to their respective mean normal times were calculated. Segregation of LA patients from all remaining patients except heparinized ones could be made with a high degree of accuracy. A thrombin time was needed to separate LA from heparinized patients. The method was highly reproducible and only minor (negligible) differences in potencies were observed between different vesicle preparations. Both the intra-batch and the inter-batch coefficients of variations on the KCTves were lower than 6%.     

A pilot randomised controlled trial comparing reactive air and active alternating pressure mattresses in the prevention and treatment of pressure ulcers among medical ICU patients

BackgroundData on the prevention and treatment of pressure ulcers (PU's) among ICU patients is sparse.ObjectiveTo compare PU outcomes in medial ICU patients nursed on either a reactive mattress overlay (ROHO^®, ROHO Inc, Belleville, IL, USA) or an active alternating pressure mattress (NIMBUS^®3, ArjoHuntleigh, Luton Bedfordshire, UK).DesignPilot prospective single blind randomised controlled clinical trial.InterventionTwo types of pressure redistributing mattress.PatientsTwo groups of eight patients.MethodsPatients included in the study were those at high risk (Norton scale <8) or with a PU on admission.ResultsThe two groups had similar patient characteristics. However, the NIMBUS 3 group presented with more ulcers per patient on admission (62.5%) and more severe ulcers (20% category 3) while four patients (50%) presented with a single superficial ulcer in the ROHO group.HealingThe progress of the ulcers showed significant decreases in PU surface area (p = 0.05), total PUSH tool score (p = 0.01) in the NIMBUS 3 group compared to the ROHO group. In the NIMBUS 3 group 82% of the ulcers improved versus none in the ROHO group (p = 0.002) and 18% remained stable versus 33%. None of the ulcers deteriorated in the NIMBUS 3 group versus 67% in the ROHO group (p = 0.006). Full thickness wounds (Category 3) were present in 22% of the NIMBUS 3 group versus 0% of the ROHO group on admission and in 0% versus 66.7% (p = 0.008) respectively at the end of the pilot study.PreventionNon-blanching erythema occurred equally in both arms at baseline; skin remained intact for the NIMBUS 3 group while 50% in the ROHO group worsened with superficial tissue loss.ConclusionThis small pilot study suggests that ‘active’ alternating therapy is a useful adjunct in the care of highly vulnerable patients, while the outcomes may be less favourable when using ‘reactive’, constant low pressure devices.     

Tuberculous meningitis: does lowering the treatment threshold result in many more treated patients?

Objective  To determine how many more patients would be treated when lowering the treatment threshold for tuberculous meningitis.
Methods  From 1989 to 2004 findings of patients with symptoms lasting more than 1 week and inflammatory changes of cerebrospinal fluid (CSF) were collected. Several models of latent class analysis were tested. Cumulative numbers of cases were plotted against different cut-offs for post-test probability.
Results  In a cohort of 232 patients the prevalence of tuberculous meningitis (TBM) was estimated at 79.8% (95% CI. 67,0–88,1); probabilities above 80% were reached in 73% of patients. Lowering this threshold from 80% to 20% would add 14% more patients to be treated, for a total of 87%. A further lowering of the threshold to 5% would imply 5% more patients to be treated, bringing the cumulative number to 92%. The difference of lowering the threshold from 80% to 5% was 19%.
Conclusion  In this setting, at least 75% of patients showing suggestive symptoms for more than a week and CSF changes very probably had TBM. The number of patients that should be treated does not increase linearly when lowering the threshold.     

Sexual risk behavior of travelers who consulted a pretravel clinic

Objective.  The objective of this study was to determine to which degree travelers who received pretravel advice at a travel clinic have protected or unprotected sexual contact with a new partner and what factors influence this behavior.
Method.  An anonymous questionnaire was sent to travelers who came to a pretravel clinic between June 1 and August 31, 2005. Risk factors for casual travel sex and predictors of protected sex were studied in a multivariate model.
Results.  A total of 1,907 travelers were included (response rate 55%) in the study. Only 4.7% of the respondents had sexual contact with a new partner, and 63.1% of these new partners were from the country of destination. Of those who had casual travel sex, 52.4% did not expect this (women 75%), 30.9% did not always use condoms, and 41% were not protected against hepatitis B. Independent risk factors for casual travel sex were traveling without steady partner (OR 14.4), expecting casual travel sex (OR 9.2), having casual sexual contacts in the home country (OR 2.4), non-tourist journeys (OR 2.2), being male (OR 2.1), the fact that the information on sexually transmitted infections (STI) had been read (OR 2.0), and traveling to South and Central America (OR 2.0). Taking condoms along (OR 5.4) and reading the information on STI (OR 3.3) were identified as independent predictors of protected sex.
Conclusions.  Travelers have substantial sexual risk behavior. Casual sex is usually not expected, and the most important predictor is traveling without a steady partner. We would advice every client of a travel clinic who will travel without a steady partner to read the STI information, to take condoms along, and to be vaccinated against hepatitis B.     

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3′ UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes.Rapid gains in DNA synthesis technologies have created new capabilities for understanding the structure, function, and evolution of genomes. Pioneering work established that native genomic DNA can be functionally replaced by synthetic DNA molecules encoding identical sequences. Milestones include the 7.5-kb synthetic poliovirus in 2002 (Cello et al. 2002), the 5.4-kb synthetic φX174 phage in 2003 (Smith et al. 2003), and the 1.1 Mbp “synthia” Mycoplasma genitalium genome in 2008 (Gibson et al. 2008). Synthetic genomes offer the possibility of redesign to improve their value for research and engineering applications, notably the full synthesis of the 40-kb refactored T7 phage in 2005 (Chan et al. 2005) and the genome-scale editing of Escherichia coli MG1655 to recode all UAG stop codons to UAA (Lajoie et al. 2013).Eukaryotic synthetic genomics has centered on Saccharomyces cerevisiae (yeast), simultaneously a powerful model organism and a producer of valuable products. Yeast genome synthesis provides access to genome biology relating to features such as chromatin structure, splicing, and linear chromosomes with telomeres, centromeres, and recombinations that are absent from prokaryotes. With the goal of answering these questions, the Sc2.0 Project has designed and synthesized synthetic chromosomes that function in yeast (Dymond et al. 2011; Annaluru et al. 2014)Beyond recapitulating native biology with a synthetic DNA molecule that is an exact copy of a natural sequence, synthetic genomics afford the possibility of designer genome features that can be exploited to learn biology and introduce valuable new capabilities. Yeast chromosomes designed as part of the Sc2.0 Project include designed site-specific recombination targets, termed loxPsym sites, which are substrates for an inducible form of the appropriate site-specific recombinase, Cre-EBD (Lindstrom and Gottschling 2009). Unlike the native directional loxP site, which permits a single orientation for recombination, the synthetic loxPsym site''s symmetry ensures that any pair of sites can recombine in either orientation (Hoess et al. 1986). Controlled expression of Cre-EBD may then lead to stochastic rearrangements of chromosome segments flanked by loxPsym sites, with deletions and inversions in principle equally likely based on the relative orientation of the loxPsym sites in the recombination junction.Previous work demonstrated the ability of this system, synthetic chromosome rearrangement and modification by loxPsym-mediated evolution (SCRaMbLE), to generate phenotype diversity by application to synIXR, a circular synthetic chromosome based on the right arm of yeast Chromosome IX. Strains generated by SCRaMbLE had heterogeneous growth rates, and auxotrophic mutants corresponded to deletions of specific loxPsym-flanked regions containing known candidate genes. Deletions provide a route for a population of cells to explore condition-specific minimal genomes, a complementary approach to expert-inferred or designed minimal genomes (Koonin 2000; Suzuki et al. 2015). Inversions and more complicated rearrangements could provide selectable diversity for rapid directed evolution.While initial applications of SCRaMbLE have been promising (Dymond and Boeke 2012), many potential challenges exist. First, introducing multiple loxPsym sites may create genome instability through homologous recombination even in the absence of Cre recombinase; subsequent to the end of Cre induction, leaky expression or continuing protein activity may lead to instability. When Cre is active, ectopic recombinations may involve “off-target” or cryptic sites in the yeast genome, outside the designed loxPsym sites; Cre generates ectopic recombinations between loxP sites and off-target sites, albeit at extremely low frequency (Sauer 1992). For desired recombinations at loxPsym sites, random pairing is desirable for maximum diversity; beyond the 82-bp minimum distance required for loxP recombination (Hoess et al. 1985), recombination hotpots may reduce the diversity. Detailed characterization of the genomes of SCRaMbLE strains are required to answer these questions, but even here the genome rearrangements generated by SCRaMbLE may not be amenable to standard genome sequencing and assembly methods.Here we test these crucial hypotheses. As described below, we sequenced the genomes of 64 synIXR SCRaMbLE strains, including the nonsynthetic as well as synthetic chromosomes to detect ectopic recombinations; we also examined genome stability for different Cre induction systems. We characterized in detail the types of recombinations detected, including deletions, inversions, and a surprisingly high frequency of duplications. Our results verify the utility of SCRaMbLE to generate combinatorial diversity on demand.