In situ formation of ligand 2,2'-[(E)-diazene-1,2-diyldicarbonothioyl]diphenol and structural characterization of its binuclear rhodium(V) complex containing RhO2+

The ligand 2,2'-[(E)-diazene-1,2-diyldicarbonothioyl]diphenol has been synthesised in situ by aerial oxidation of o-hydroxythiobenzhydrazide [H(htbh)] in presence of rhodium(III) in DMSO. Each ligand binds two RhO2+ ions through its N and S atoms and the O atom of its deprotonated hydroxy group. Each RhO2+ contains two cis-Rh = O bonds. The sixth coordination site of each rhodium(v) is occupied by the O of DMSO.

A flow system for the on-line quantitative measurement of the retention of dosage forms on biological surfaces using spectroscopy and image analysis

Measuring the retention, or residence time, of dosage forms to biological tissue is commonly a qualitative measurement, where no real values to describe the retention can be recorded. The result of this is an assessment that is dependent upon a user's interpretation of visual observation. This research paper outlines the development of a methodology to quantitatively measure, both by image analysis and by spectrophotometric techniques, the retention of material to biological tissues, using the retention of polymer solutions to ocular tissue as an example. Both methods have been shown to be repeatable, with the spectrophotometric measurement generating data reliably and quickly for further analysis.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella entetica serovar typhimurium selected following exposure to disinfectants

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-To1C for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

Local orientational disorder in peptide fibrils probed by a combination of residue-specific 13C-18O labelling, polarised infrared spectroscopy and molecular combing

A novel combination of site-specific isotope labelling, polarised infrared spectroscopy and molecular combing reveal local orientational ordering in the fibril-forming peptide YTIAALLSPYSGGRADS. Use of 13C-18O labelled alanine residues demonstrates that the Nterminal end of the peptide is incorporated into the cross-beta structure, while the C-terminal end shows orientational disorder

Provenance of marbles from Naxos based on microstructural and geochemical characterization

A detailed quantitative microstructural study coupled with cathodoluminescence and geochemical analyses on marbles from Naxos demonstrates that the analysis of microstructures is the most sensitive method to define the origin of marbles within, and between, different regions. Microstructure examination can only be used as an accurate provenance tool if a correction for the second-phase content is considered. If second phases are not considered, a large spread of different microstructures occurs within sample sites, making a separation between neighbouring outcrops difficult or impossible. Moreover, this study shows that the origin of a marble is defined more precisely if the microstructural observations are coupled with cathodoluminescence data.

Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)8–13 expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

Is optical imaging spectroscopy a viable measurement technique for the investigation of the negative BOLD phenomenon? A concurrent optical imaging spectroscopy and fMRI study at high field (7T)

Traditionally functional magnetic resonance imaging (fMRI) has been used to map activity in the human brain by measuring increases in the Blood Oxygenation Level Dependent (BOLD) signal. Often accompanying positive BOLD fMRI signal changes are sustained negative signal changes. Previous studies investigating the neurovascular coupling mechanisms of the negative BOLD phenomenon have used concurrent 2D-optical imaging spectroscopy (2D-OIS) and electrophysiology (Boorman et al., 2010). These experiments suggested that the negative BOLD signal in response to whisker stimulation was a result of an increase in deoxy-haemoglobin and reduced multi-unit activity in the deep cortical layers. However, Boorman et al. (2010) did not measure the BOLD and haemodynamic response concurrently and so could not quantitatively compare either the spatial maps or the 2D-OIS and fMRI time series directly. Furthermore their study utilised a homogeneous tissue model in which is predominantly sensitive to haemodynamic changes in more superficial layers. Here we test whether the 2D-OIS technique is appropriate for studies of negative BOLD. We used concurrent fMRI with 2D-OIS techniques for the investigation of the haemodynamics underlying the negative BOLD at 7 Tesla. We investigated whether optical methods could be used to accurately map and measure the negative BOLD phenomenon by using 2D-OIS haemodynamic data to derive predictions from a biophysical model of BOLD signal changes. We showed that despite the deep cortical origin of the negative BOLD response, if an appropriate heterogeneous tissue model is used in the spectroscopic analysis then 2D-OIS can be used to investigate the negative BOLD phenomenon.

Dynamics and diversity of the 'Atopobium cluster' in the human faecal microbiota, and phenotypic characterization of 'Atopobium cluster' isolates

This study monitored the dynamics and diversity of the human faecal 'Atopobium cluster' over a 3-month period using a polyphasic approach. Fresh faecal samples were collected fortnightly from 13 healthy donors (6 males and 7 females) aged between 26 and 61 years. Fluorescence in situ hybridization was used to enumerate total (EUB338mix) and 'Atopobium cluster' (ATO291) bacteria, with counts ranging between 1.12 × 1011 and 9.95 × 1011, and 1.03 × 109 and 1.16 × 1011 cells (g dry weight faeces)-1, respectively. The 'Atopobium cluster' population represented 0.2-22 % of the total bacteria, with proportions donor-dependent. Denaturing gradient gel electrophoresis (DGGE) using 'Atopobium cluster'-specific primers demonstrated faecal populations of these bacteria were relatively stable, with bands identified as Collinsella aerofaciens, Collinsella intestinalis/Collinsella stercoris, Collinsella tanakaei, Coriobacteriaceae sp. PEAV3-3, Eggerthella lenta, Gordonibacter pamelaeae, Olsenella profusa, Olsenella uli and Paraeggerthella hongkongensis in the DGGE profiles of individuals. Colony PCR was used to identify 'Atopobium cluster' bacteria isolated from faeces (n = 224 isolates). 16S rRNA gene sequence analysis of isolates demonstrated Collinsella aerofaciens represented the predominant (88 % of isolates) member of the 'Atopobium cluster' found in human faeces, being found in nine individuals. Eggerthella lenta was identified in three individuals (3.6 % of isolates). Isolates of Collinsella tanakaei, an 'Enorma' sp. and representatives of novel species belonging to the 'Atopobium cluster' were also identified in the study. Phenotypic characterization of the isolates demonstrated their highly saccharolytic nature and heterogeneous phenotypic profiles, and 97 % of the isolates displayed lipase activity.

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

New insights into the compressibility and high-pressure stability of Ni(CN)2: a combined study of neutron diffraction, Raman spectroscopy, and inelastic neutron scattering

Nickel cyanide is a layered material showing markedly anisotropic behaviour. High-pressure neutron diffraction measurements show that at pressures up to 20.1 kbar, compressibility is much higher in the direction perpendicular to the layers, c, than in the plane of the strongly chemically bonded metal-cyanide sheets. Detailed examination of the behaviour of the tetragonal lattice parameters, a and c, as a function of pressure reveal regions in which large changes in slope occur, for example, in c(P) at 1 kbar. The experimental pressure dependence of the volume data is fitted to a bulk modulus, B0, of 1050 (20) kbar over the pressure range 0–1 kbar, and to 124 (2) kbar over the range 1–20.1 kbar. Raman spectroscopy measurements yield additional information on how the structure and bonding in the Ni(CN)2 layers change with pressure and show that a phase change occurs at about 1 kbar. The new high-pressure phase, (Phase PII), has ordered cyanide groups with sheets of D4h symmetry containing Ni(CN)4 and Ni(NC)4 groups. The Raman spectrum of phase PII closely resembles that of the related layered compound, Cu1/2Ni1/2(CN)2, which has previously been shown to contain ordered C≡N groups. The phase change, PI to PII, is also observed in inelastic neutron scattering studies which show significant changes occurring in the phonon spectra as the pressure is raised from 0.3 to 1.5 kbar. These changes reflect the large reduction in the interlayer spacing which occurs as Phase PI transforms to Phase PII and the consequent increase in difficulty for out-of-plane atomic motions. Unlike other cyanide materials e.g. Zn(CN)2 and Ag3Co(CN)6, which show an amorphization and/or a decomposition at much lower pressures (~100 kbar), Ni(CN)2 can be recovered after pressurising to 200 kbar, albeit in a more ordered form.

Genetic and phenotypic characterization of complex hereditary spastic paraplegia

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease- causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15. Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson’s disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.

Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta)

Species of Gracilaria are some of the most useful algae in the world for the production of agar. As a consequence of its economic importance, the genus has been the subject of many studies worldwide. Color variants of Gracilaria birdiae have been found in the natural population on the Brazilian coast, and they have also been isolated from plants cultivated in laboratory. These findings raised new questions regarding intraspecific variation and the prospects of cultivating such variants for their agar production. Therefore, this work aimed to determine the mode of color inheritance for two G. birdiae strains: a greenish-brown strain (gb) found in a natural population and a green strain (gr) which had arisen as a spontaneous mutation in a red plant cultured in the laboratory. The pigment contents of these strains, as well as the red wildtype (rd), were also characterized. Crosses between female and male plants of the same color (rd, gr, or gb) and between different colors were performed. Crosses between plants of the same color showed tetrasporophytic and gametophytic descendents of the parental color. Recessive nuclear inheritance was found in the greenish-brown strain, and cytoplasmic maternal inheritance was found in the green strain; both had lower phycoerythrin and higher concentrations of allophycocyanin and phycocyanin than the wild-type. Chlorophyll a contents were similar among all strains. Taken together, our results contribute to knowledge about the variability of this important red algae. In addition, since greenish-brown and green strains showed stability of color, both could be selected and tested in experimental sea cultivation to evaluate if mutants have advantageous performance when compared with red strain.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM AND HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.