Potentiation of antiseizure and neuroprotective efficacy of standard nerve agent treatment by addition of tariquidar

Organophosphate (OP) induced seizures are commonly treated with anticholinergics, oximes and anticonvulsants. Inhibition of P-glycoprotein (PgP) has been shown to enhance the efficacy of nerve agent treatment in soman exposed rats. In the present study, the promising effects of the PgP inhibitor tariquidar were investigated in more detail in rats s.c. exposed to 150 μg/kg soman. Treatment with HI-6 and atropine sulfate (125 and 3 mg/kg i.m respectively) was administered 1 min after exposure. Diazepam (0.5 mg/kg i.m.) and/or tariquidar (7.5 mg/kg i.v.) were included either at 1 min or 40 min following onset of seizures. Animals that received tariquidar, in addition to HI-6 and atropine, at 1 min, displayed a rapid normalization of EEG activity and cessation of seizure-associated behaviour. This improvement by addition of tariquidar was even more substantial in animals that also received diazepam, either immediately or delayed. Animals exhibiting lower intensity seizures displayed less severe neuropathology (neuronal loss, microglia activation and astrogliosis), primarily in the piriform cortex, and to a lesser extent amygdala and entorhinal cortex. The present findings suggest that the interaction of tariquidar with atropine may be the decisive factor for enhanced treatment efficacy, given that atropine was previously found to be a PgP substrate. A more thorough understanding of the interactions of nerve agent antidotes, in particular the actions of central anticholinergics with benzodiazepines, could contribute to a future optimization of treatment combinations, particularly those aimed at later stage medical interventions

Ultrafast Structural Dynamics of Photo-Reactions Revealed by Model-Independent X-ray Cross-Correlation Analysis

We applied angular X-ray Cross-Correlation analysis (XCCA) to scattering images from a femtosecond resolution LCLS X-ray free-electron laser (XFEL) pump-probe experiment with solvated PtPOP ([Pt$_2$(P$_2$O$_5$H$_2$)$_4$]$^{4-}$) metal complex molecules. The molecules were pumped with linear polarized laser pulses creating an excited state population with a preferred orientational (alignment) direction. Two time scales of $1.9\pm1.5$ ps and $46\pm10$ ps were revealed by model-independent XCCA, associated with an internal structural changes and rotational dephasing, respectively. Our studies illustrate the potential of XCCA to reveal hidden structural information in a model independent analysis of time evolution of solvated metal complex molecules.Comment: 8 pages, 5 figures, 50 reference

Long range allostery mediates cooperative adenine nucleotide binding by the Ski2 like RNA helicase Brr2

Brr2 is an essential Ski2 like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N terminal helicase cassette and a structurally similar but enzymatically inactive C terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C terminal cassette is more than two orders of magnitude higher than that of the active N terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C terminal cassette reduces nucleotide binding at the N terminal cassette 70 away. Molecular dynamics simulations identified structural communication lines that likely mediate these long range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicin

Chronic care for heart failure patients: who to refer back to the general practitioner?: Experiences of the Dutch integrated heart failure care model

ObjectiveThe number of patients with heart failure (HF) and corresponding burden of the healthcare system will increase significantly. The Dutch integrated model, 'Transmural care of HF Patients' was based on the European Society of Cardiology (ESC) guidelines and initiated to manage the increasing prevalence of HF patients in primary and secondary care and stimulate integrated care. It is unknown how many HF patients are eligible for back-referral to general practitioners (GPs), which is important information for the management of chronic HF care. This study aims to evaluate clinical practice of patients for whom chronic HF care can be referred from the cardiologist to the GP based on the aforementioned chronic HF care model.Design and MethodsA retrospective case record-based study was conducted, which included all chronic HF patients registered in the cardiology information systems of two different hospitals. Subsequently, 200 patients were randomly selected for evaluation. The following patients were considered eligible for referral to the GP: 1/Stable HF patients with reduced left ventricular ejection fraction (LVEF), 2/Stable HF patients with a recovered LVEF and 3/Stable HF patients with a preserved LVEF, 4/HF, palliative setting.ResultsOf the 200 patients, 17% was considered eligible for referral to the GP. This group consisted of 5% patients with a reduced LVEF, 10.5% patients with recovered LVEF and 1.5% patients with a preserved LVEF. Main indicators for HF care by cardiologists were active cardiac disease other than HF (39.5%), recent admission for HF (29.5%) or a recent adjustment in HF medication (7.5%).ConclusionApplying the chronic HF care model of the 'Transmural care of HF patients' and the ESC-guidelines, results in an important opportunity to further optimise HF integrated care and to deal with the increasing number of HF patients referred to the hospital

The crystal structure of an ‘All Locked’ nucleic acid duplex

‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern

Site-specific O-glycosylation on the MUC_{2} mucin protein inhibits cleavage by the porphyromonas gingivalis secreted cysteine protease (RgpB)

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation