Rapid species and strain differentiation of non-tubercoulous mycobacteria by Fourier-Transform Infrared microspectroscopy

Rapid identification of non-tuberculous mycobacteria (NTM) species is important in clinical laboratories to stipulate the appropriate therapy and to offer a comprehensive infection control. We applied Fourier-Transform Infrared microspectroscopy to evaluate, whether the most frequent species of NTM can be rapidly and uniformly identified by this method using microcolonies of NTM growing on solid nutrient agar plates. To establish a standardized protocol, the heterogeneity of cell growth within the microcolonies and the reproducibility of measuring the IR spectra from whole mycobacterial microcolonies were first studied. Hierarchical cluster analysis applied to spectra obtained by linear mapping across microcolony imprints from fast- and slow-growing NTM revealed only little spectral variance between the various microcolony zones. In parallel, when repetitive measurements were performed on independently grown whole single microcolonies with diameters of 80 and 140 mum, excellent reproducibility could be achieved, verifying that mycobacterial microcolonies are well suited for FT-IR-based identification. Twenty-eight different and well-defined strains, comprising the most frequent species of NTM isolated in clinical laboratories, were used to create a classification system based on FT-IR spectra from single microcolonies. Hierarchical cluster analysis allowed the assignment of all isolates measured in replicates to their correct species-specific clusters. Additionally, a clear separation of all strains into strain-specific sub-clusters was observed. These results demonstrate the potential of FT-IR microspectroscopy to rapidly differentiate NTM at the species and strain level. The data so far obtained suggest that an extended spectral database, containing more NTM strains and covering a broader biological variance, may provide a practical solution to rapidly identify unknown NTM isolates in routine clinical-microbiological laboratories with the additional possibility to type these microorganisms at the sub-species level.     

Formation and properties of chitosan-cellulose nanocrystal polyelectrolyte-macroion complexes for drug delivery applications

This study examines a novel polyelectrolyte-macroion complex (PMC) between chitosan, a cationic polysaccharide, and cellulose nanocrystals (CNCs), anionic, cylindrical nanoparticles, for potential applications in drug delivery. CNCs were prepared by H(2)SO(4) hydrolysis of wood pulp. The formation of PMCs was monitored by turbidimetric titration. In titrations of a chitosan solution with a CNC suspension, the turbidity reached a plateau, but it had a maximum and then decreased when the direction of titration was reversed. PMC particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, and laser Doppler electrophoresis. The particles were composed primarily of CNCs and ranged in size from a few hundred nanometers to several micrometers, depending on the cellulose/chitosan ratio. Particles formed at amino/sulfate group molar ratios >1 were nearly spherical in shape and positively charged, whereas particles formed at ratios <1 had well-defined nonspherical shapes and were negatively charged.     

Exclusion of the C/D box snoRNA gene cluster HBII-52 from a major role in Prader–Willi syndrome

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurogenetic disorders caused by the loss of function of imprinted genes in 15q11–q13. The maternally expressed UBE3A gene is affected in AS. Four protein-encoding genes (MKRN3, MAGEL2, NDN and SNURF-SNRPN) and several small nucleolar (sno) RNA genes (HBII-13, HBII-436, HBII-85, HBII-438A, HBII-438B and HBII-52) are expressed from the paternal chromosome only but their contribution to PWS is unclear. To examine the role of the HBII-52 snoRNA genes, we have reinvestigated an AS family with a submicroscopic deletion spanning UBE3A and flanking sequences. By fine mapping of the centromeric deletion breakpoint in this family, we have found that the deletion affects all of the 47 HBII-52 genes. Since the complete loss of the HBII-52 genes in family members who carry the deletion on their paternal chromosome is not associated with an obvious clinical phenotype, we conclude that HBII-52 snoRNA genes do not play a major role in PWS. However, we cannot exclude the possibility that the loss of HBII-52 has a phenotypic effect when accompanied by the loss of function of other genes in 15q11–q13.Electronic Database Information: accession numbers and URLs for data presented herein are as follows: for PAR-4 (accession number AF019617), deletion junction fragment (L15422): GenBank, ; for Angelman syndrome [MIM105830]: Online Mendelian Inheritance in Man (OMIM),     

Structural and functional diversities among mu-conotoxins targeting TTX-resistant sodium channels

mu-Conotoxins are peptides that block sodium channels. Molecular cloning was used to identify four novel mu-conotoxins: CnIIIA, CnIIIB, CIIIA, and MIIIA from Conus consors, C. catus and C. magus. A comparison of their sequences with those of previously characterized mu-conotoxins suggested that the new mu-conotoxins were likely to target tetrodotoxin-resistant (TTX-r) sodium channels. The four peptides were chemically synthesized, and their biological activities were characterized. The new conotoxins all blocked, albeit with varying potencies, TTX-r sodium currents in frog dorsal-root-ganglion (DRG) neurons. The more potent of the four new mu-conotoxins, CnIIIA and CIIIA, exhibited a strikingly different selectivity profile in blocking TTX-r versus TTX-sensitive channels, as determined by their ability to block extracellularly recorded action potentials in three preparations from frog: skeletal muscle, cardiac muscle and TTX-treated C-fibers. CnIIIA was highly specific for TTX-r sodium channels, whereas CIIIA was nonselective. Both peptides appeared significantly less potent in blocking TTX-r sodium currents in rat and mouse DRG neurons. When CnIIIA and CIIIA were injected intracranially into mice, both induced seizures, but only CIIIA caused paralysis. This is the most comprehensive characterization to date of the structural and functional diversities of an emerging group of mu-conotoxins targeting TTX-r sodium channels.     

Mutated mtDNA Distribution in Exponentially Growing Cell Cultures and How the Segregation Rate is Increased by the Mitochondrial Compartments

A cell contains many copies of mitochondrial DNA. The distribution of a mitochondrial gene mutation in a cell culture is governed by the way in which the mtDNA molecules of a cell are replicated and partitioned between the two daughter cells during mitosis. Assuming that this partition process is random, we describe the evolution of the mitochondrial genetic state of a cell culture. The mutated mtDNA is ultimately segregated and the rate of the trend to segregation is relatively slow. It is nevertheless greatly accelerated if the model takes into account the spatial mtDNA partition by the mitochondrial compartments.     

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein–protein interactions with other regulators such as phytochrome (FphA) or velvet‐like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi‐phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild‐type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V‐T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.