Taming tosyl azide: the development of a scalable continuous diazo transfer process

Heat and shock sensitive tosyl azide was generated and used on demand in a telescoped diazo transfer process. Small quantities of tosyl azide were accessed in a one pot batch procedure using shelf stable, readily available reagents. For large scale diazo transfer reactions tosyl azide was generated and used in a telescoped flow process, to mitigate the risks associated with handling potentially explosive reagents on scale. The in situ formed tosyl azide was used to rapidly perform diazo transfer to a range of acceptors, including β-ketoesters, β-ketoamides, malonate esters and β-ketosulfones. An effective in-line quench of sulfonyl azides was also developed, whereby a sacrificial acceptor molecule ensured complete consumption of any residual hazardous diazo transfer reagent. The telescoped diazo transfer process with in-line quenching was used to safely prepare over 21 g of an α-diazocarbonyl in >98% purity without any column chromatography

Haemophilia care in Europe:a survey of 19 countries

In 2009, a questionnaire was circulated to 19 national haemophilia patient organizations in Europe affiliated to the European Haemophilia Consortium (EHC) and the World Federation of Hemophilia (WFH) to seek information about the organization of haemophilia care and treatment available at a national level. The responses received highlighted differences in the level of care despite the recent promulgation of consensus guidelines designed to standardize the care of haemophilia throughout the continent of Europe. There was a wide range in factor VIII consumption with usage ranging from 0.38 IU per capita in Romania to 8.7 IU per capita in Sweden (median: 3.6 IU per capita). Despite the specific inclusion of coagulation factor concentrate in the WHO list of essential medications, cryoprecipitate is still used in some eastern European countrie

Estrous cycle influences excitatory amino acid transport and visceral pain sensitivity in the rat: Effects of early-life stress

Background: Early-life stress (ELS) is a recognized risk factor for chronic pain disorders, and females appear to be more sensitive to the negative effects of stress. Moreover, estrous cycle-related fluctuations in estrogen levels have been linked with alternating pain sensitivity. Aberrant central circuitry involving both the anterior cingulate cortex (ACC) and the lumbosacral spinal cord has also been implicated in the modulation of visceral pain in clinical and preclinical studies. Here we further investigate changes in visceral pain sensitivity and central glutamatergic systems in rats with respect to estrous cycle and ELS. Methods: We investigated visceral sensitivity in adult female Sprague-Dawley rats, which had undergone maternal separation (MS) in early life or remained non-separated (NS), by performing colorectal distension (CRD). We also assessed excitatory amino acid uptake through excitatory amino acid transporters (EAATs) in the lumbosacral spinal cord and ACC. Results: NS animals in proestrus and estrus exhibited reduced EAAT uptake and decreased threshold to CRD. Moreover, total pain behaviors were increased in these stages. MS rats exhibited lower pain thresholds and higher total pain behaviors to CRD across all stages of the estrous cycle. Interestingly, cortical EAAT function in MS rats was inhibited in the low estrogen state—an effect completely opposite to that seen in NS rats. Conclusions: This data confirms that estrous cycle and ELS are significant factors in visceral sensitivity and fluctuations in EAAT function may be a perpetuating factor mediating central sensitization

Characterisation of cationic amphiphilic cyclodextrins for neuronal delivery of siRNA: effect of reversing primary and secondary face modifications

Significant research is focused on the development of non-viral vectors for delivery of siRNA to neurons and the central nervous system. Cyclodextrins (CDs) have shown great promise as efficient and low toxicity gene delivery vectors in various cell types. Here, we investigate two CDs for siRNA delivery in a neuronal cell model. These CDs were substituted on opposite faces (primary and secondary) with amphiphilic and cationic groups. Physical properties of CD.siRNA complexes, including size, charge and stability were measured. In vitro investigations were carried out in immortalised hypothalamic neurons. Neuronal cell uptake was measured by flow cytometry and cytotoxicity was assessed by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Both CDs interacted with siRNA, yielding nanosized cationic complexes which exhibited good stability on storage. A favourable toxicity profile was demonstrated for the CD.siRNA complexes. However, only one of the two CDs mediated high levels of neuronal uptake and efficient gene silencing, equivalent to those achieved with a commercial lipid-based vector. Despite the suitability of both CDs as siRNA delivery vectors in terms of their ability to complex siRNA and the properties of the complexes yielded, only one CD achieved good transfection efficiency. This was likely due to the differences in their chemical structures. The effective CD offers great potential as a novel non-toxic vector for neuronal siRNA delivery

The rotation and coma profiles of comet C/2004 Q2 (Machholz)

Aims. Rotation periods of cometary nuclei are scarce, though important when studying the nature and origin of these objects. Our aim is to derive a rotation period for the nucleus of comet C/2004 Q2 (Machholz). Methods. C/2004 Q2 (Machholz) was monitored using the Merope CCD camera on the Mercator telescope at La Palma, Spain, in January 2005, during its closest approach to Earth, implying a high spatial resolution (50km per pixel). One hundred seventy images were recorded in three different photometric broadband filters, two blue ones (Geneva U and B) and one red (Cousins I). Magnitudes for the comet's optocentre were derived with very small apertures to isolate the contribution of the nucleus to the bright coma, including correction for the seeing. Our CCD photometry also permitted us to study the coma profile of the inner coma in the different bands. Results. A rotation period for the nucleus of P = 9.1 +/- 0.2 h was derived. The period is on the short side compared to published periods of other comets, but still shorter periods are known. Nevertheless, comparing our results with images obtained in the narrowband CN filter, the possibility that our method sampled P/2 instead of P cannot be excluded. Coma profiles are also presented, and a terminal ejection velocity of the grains v_gr = 1609 +/- 48 m/s is found from the continuum profile in the I band.Comment: 11 pages, 9 figures, accepted by A&

Inflammation and Immune-Related Candidate Gene Associations with Acute Lung Injury Susceptibility and Severity: A Validation Study

Introduction: Common variants in genes related to inflammation, innate immunity, epithelial cell function, and angiogenesis have been reported to be associated with risks for Acute Lung Injury (ALI) and related outcomes. We tested whether previously-reported associations can be validated in an independent cohort at risk for ALI. Methods: We identified 37 genetic variants in 27 genes previously associated with ALI and related outcomes. We prepared allelic discrimination assays for 12 SNPs from 11 genes with MAF>0.05 and genotyped these SNPs in Caucasian subjects from a cohort of critically ill patients meeting criteria for the systemic inflammatory response syndrome (SIRS) followed for development of ALI, duration of mechanical ventilation, and in-hospital death. We tested for associations using additive and recessive genetic models. Results: Among Caucasian subjects with SIRS (n = 750), we identified a nominal association between rs2069832 in IL6 and ALI susceptibility (OR$_{adj}$ 1.61; 95% confidence interval [CI], 1.04–2.48, P = 0.03). In a sensitivity analysis limiting ALI cases to those who qualified for the Acute Respiratory Distress Syndrome (ARDS), rs61330082 in NAMPT was nominally associated with risk for ARDS. In terms of ALI outcomes, SNPs in MBL2 (rs1800450) and IL8 (rs4073) were nominally associated with fewer ventilator-free days (VFDs), and SNPs in NFE2L2 (rs6721961) and NAMPT (rs61330082) were nominally associated with 28-day mortality. The directions of effect for these nominal associations were in the same direction as previously reported but none of the associations survived correction for multiple hypothesis testing. Conclusion: Although our primary analyses failed to statistically validate prior associations, our results provide some support for associations between SNPs in IL6 and NAMPT and risk for development of lung injury and for SNPs in IL8, MBL2, NFE2L2 and NAMPT with severity in ALI outcomes. These associations provide further evidence that genetic factors in genes related to immunity and inflammation contribute to ALI pathogenesis

Click-modified cyclodextrins as non-viral vectors for neuronal siRNA delivery

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors, which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified β-cyclodextrin (CD) vector was synthesised, which complexed siRNA to form cationic nanoparticles of less than 200nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalised hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labelled siRNA, whilst maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells

Sturmian bases for two-electron systems in hyperspherical coordinates

We give a detailed account of an ab initio spectral approach for the calculation of energy spectra of two active electron atoms in a system of hyperspherical coordinates. In this system of coordinates, the Hamiltonian has the same structure as the one of atomic hydrogen with the Coulomb potential expressed in terms of a hyperradius and the nuclear charge replaced by an angle dependent effective charge. The simplest spectral approach consists in expanding the hyperangular wave function in a basis of hyperspherical harmonics. This expansion however, is known to be very slowly converging. Instead, we introduce new hyperangular Sturmian functions. These functions do not have an analytical expression but they treat the first term of the multipole expansion of the electron–electron interaction potential, namely the radial electron correlation, exactly. The properties of these new functions are discussed in detail. For the basis functions of the hyperradius, several choices are possible. In the present case, we use Coulomb–Sturmian functions of half integer angular momentum. We show that, in the case of H−, the accuracy of the energy and the width of the resonance states obtained through a single diagonalization of the Hamiltonian, is comparable to the values given by state-of-the-art methods while using a much smaller basis set. In addition, we show that precise values of the electric-dipole oscillator strengths for ${\rm{S}}\to {\rm{P}}$ transitions in helium are obtained thereby confirming the accuracy of the bound state wave functions generated with the present method