In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB,katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

Benefits of Turbid River Plume Habitat for Lake Erie Yellow Perch (Perca flavescens) Recruitment Determined by Juvenile to Larval Genotype Assignment

Nutrient-rich, turbid river plumes that are common to large lakes and coastal marine ecosystems have been hypothesized to benefit survival of fish during early life stages by increasing food availability and (or) reducing vulnerability to visual predators. However, evidence that river plumes truly benefit the recruitment process remains meager for both freshwater and marine fishes. Here, we use genotype assignment between juvenile and larval yellow perch (Perca flavescens) from western Lake Erie to estimate and compare recruitment to the age-0 juvenile stage for larvae residing inside the highly turbid, south-shore Maumee River plume versus those occupying the less turbid, more northerly Detroit River plume. Bayesian genotype assignment of a mixed assemblage of juvenile (age-0) yellow perch to putative larval source populations established that recruitment of larvae was higher from the turbid Maumee River plume than for the less turbid Detroit River plume during 2006 and 2007, but not in 2008. Our findings add to the growing evidence that turbid river plumes can indeed enhance survival of fish larvae to recruited life stages, and also demonstrate how novel population genetic analyses of early life stages can contribute to determining critical early life stage processes in the fish recruitment process.     

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90-95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α-targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α-targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.     

Chemosystematic implications of flavonoids in Aniba riparia

The trunk wood of Aniba riparia (Nees) Mez (Lauraceae) contains flavokawin-B, (2S)-pinostrombin, (2S)-5, 7-di-O-methylpinocembrin, (2R, 3R)-5, 7-di-O-methylpinobanksin, izalpinin and 3,5, 7-tri-O-methylgalangin. Structural comparison of these flavonoids with the pyrones and neolignans, which characterized all previously examined Aniba spp., leads to a chemical classification of the genus.     

The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice

Purinergic Signalling - The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate...     

Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling

Numerous studies have demonstrated that fertilization with nutrients such as nitrogen, phosphorus, and potassium increases plant productivity in both natural and managed ecosystems, demonstrating that primary productivity is nutrient limited in most terrestrial ecosystems. In contrast, it has been demonstrated that heterotrophic microbial communities in soil are primarily limited by organic carbon or energy. While this concept of contrasting limitations, that is, microbial carbon and plant nutrient limitation, is based on strong evidence that we review in this paper, it is often ignored in discussions of ecosystem response to global environment changes. The plant‐centric perspective has equated plant nutrient limitations with those of whole ecosystems, thereby ignoring the important role of the heterotrophs responsible for soil decomposition in driving ecosystem carbon storage. To truly integrate carbon and nutrient cycles in ecosystem science, we must account for the fact that while plant productivity may be nutrient limited, the secondary productivity by heterotrophic communities is inherently carbon limited. Ecosystem carbon cycling integrates the independent physiological responses of its individual components, as well as tightly coupled exchanges between autotrophs and heterotrophs. To the extent that the interacting autotrophic and heterotrophic processes are controlled by organisms that are limited by nutrient versus carbon accessibility, respectively, we propose that ecosystems by definition cannot be ‘limited’ by nutrients or carbon alone. Here, we outline how models aimed at predicting non‐steady state ecosystem responses over time can benefit from dissecting ecosystems into the organismal components and their inherent limitations to better represent plant–microbe interactions in coupled carbon and nutrient models.