Genome-wide linkage and sequence analysis challenge CCDC66 as a human retinal dystrophy candidate gene and support a distinct NMNAT1-related fundus phenotype.

To uncover the genotype underlying early-onset cone-rod dystrophy and central nummular macular atrophic lesion in 2 siblings from an endogamous Arab family, we performed targeted next-generation sequencing (NGS) of 44 retinal dystrophy genes, whole-exome sequencing (WES) and genome-wide linkage analysis. Targeted NGS and WES in the index patient highlighted 2 homozygous variants, a CCDC66 frameshift deletion and a novel missense NMNAT1 variant, c.500G>A (p.Asn167Ser). Linkage and segregation analysis excluded the CCDC66 variant and confirmed the NMNAT1 mutation. Biallelic NMNAT1 mutations cause Leber congenital amaurosis with a central nummular macular atrophic lesion (LCA9). The NMNAT1 mutation reported here underlied cone-rod dystrophy rather than LCA but the fundus lesion was compatible with that of LCA9 patients, highlighting that such a fundus appearance should raise suspicion for biallelic mutations in NMNAT1 when in the context of any retinal dystrophy. Although Ccdc66 mutations have been proposed to cause retinal disease in dogs, our results and public databases challenge CCDC66 as a candidate gene for human retinal dystrophy.

A common NYX mutation in Flemish patients with X linked CSNB.

AIMS: The Schubert-Bornschein type of complete congenital stationary night blindness (CSNB) is a genetically heterogeneous retinal disorder. It is characterised by a non-progressive disease course, often associated with high myopia and nystagmus. So far, mutations in two genes, NYX (nyctalopin) and GRM6 (metabotropic glutamate receptor 6) have been associated with this form of CSNB. The purpose of this study was to identify the genetic defect in affected male patients from Flemish families with complete CSNB. METHODS: Probands with CSNB from three large Flemish families underwent ophthalmological examination. DNA was extracted from peripheral blood, and the coding region of NYX along with parts of the 5'UTR and 3'UTR and intronic regions covering the splice sites were PCR amplified and sequenced. RESULTS: In the affected individuals of three Flemish families with the complete form of CSNB a novel NYX mutation, c.855delG was identified. This deletion is predicted to lead to a frameshift mutation, p.Asp286ThrfsX62 causing a premature stop codon. CONCLUSION: Previously, both single families with different mutations in NYX as well as different families with an identical mutation, suggestive of a founder mutation, have been described. The c.855delG deletion in NYX seems to be a common mutation associated with CSNB in the Flemish population from Belgium. Thus, we suggest performing diagnostic testing for CSNB in the Flemish population initially directed towards the identification of this mutation. Subsequent screening for other mutations in NYX or GRM6 could be performed as a second step.

Isolation of putative probionts from cod rearing environment.

Survival problems are encountered at early stages of intensive fish rearing and antibiotics are widely used to remedy the situation. Probiotics may provide a potential alternative method to protect larvae from opportunistic and pathogenic bacteria and promote a balanced environment. This study was designed to search for new probiotics to target this critical period in cod rearing. Potential probionts were selected from the natural microbiota of cod aquacultural environment. The selection was based on several criteria: pathogen inhibition potential, growth characteristics, strain identification, metabolite production and adhesion to fish cell lines. Our study demonstrated that 14% of screened bacteria (n=188) had antagonistic properties towards fish pathogens. The majority of these isolates were Gram-positive (81%), belonging to Firmicutes (69.2%) and Actinobacteria (11.5%) phyla based on 16S rRNA gene sequencing. Only 6 (3.2%) of 188 isolates could inhibit all three pathogens tested: Vibrio anguillarum, Aeromonas salmonicida subsp. achromogenes and Vibrio salmonicida. Differences observed in activity intensity and spectrum among inhibitory isolates emphasise the need to develop probiotic mixtures for efficient prophylactic methods. Comparison of growth behaviour of inhibitory isolates and pathogens at cod rearing temperatures, metabolite production and adhesion capacity were considered for final probiont selection. Four promising isolates that could be used as a mixed supplement to rearing water were identified as putative probiotic bacteria. This study emphasises the importance and potential of lactic acid bacteria in aquaculture.

Geroderma osteodysplasticum hereditaria and wrinkly skin syndrome in 22 patients from Oman.

Excessive skin wrinkling and cutis laxa are seen in many genetic conditions and overlapping features can make a clinical diagnosis difficult. Here we report on 22 Omani patients from 11 consanguineous families with the diagnosis of wrinkly skin syndrome (WSS, OMIM 278250) or geroderma osteodysplasticum hereditaria (GO, OMIM 231070). The WSS phenotype evolves during early childhood and includes a generalized and excessive skin wrinkling, dental problems, herniae, foot deformities, hip dislocations, growth retardation, and a large anterior fontanelle. The facial gestalt is characterized by a broad nasal bridge, hypertelorism, and downslanting palpebral fissures. We were unable to differentiate between WSS and cutis laxa with growth and developmental delay (CLGDD, OMIM 219200) suggesting that both can be considered as one entity. Distinct hallmarks of GO were skin wrinkling limited to the dorsum of hands and feet and to the abdomen, normal fontanelles, maxillary hypoplasia, bowed long bones, and osteopenia with frequent fractures. In contrast to the attenuation of the skin phenotype with age in WSS, adult patients with GO appeared prematurely aged. A serum sialotransferrin type 2 pattern was found in all four WSS patients tested. Apolipoprotein CIII (a marker for O-glycosylation) was normal suggesting that WSS is frequently associated with a N-protein glycosylation defect, probably at the level of processing (CDG-II). All four investigated GO patients showed normal sialotransferrin patterns. The known loci for cutis laxa and WSS on 2q31, 5q23-q31, 7q11, 11q13, and 14q32 were excluded. We suggest that WSS and GO are distinct entities with overlapping features.

Differential inactivation of Listeria monocytogenes by D- and L-lactic acid.

AIMS: To determine inactivation of Listeria monocytogenes by the two lactic acid isomers. METHODS AND RESULTS: The survival of four strains with varying sensitivity to acid was determined following treatment with L- or D-lactic acid at 100 mmol l(-1) (pH 3.7) or HCl at pH 3.37. There was some, but not complete, similarity in the relative sensitivity of the four strains to the two types of acid. All strains were most sensitive to D-lactic acid, which gave 0.6-2.2 log units greater reduction than L-lactic acid midway in the inactivation curves. Even very low concentrations of the two isomers had an immediate effect on pH(i) which was identical for the two isomers. CONCLUSIONS: The results show that L. monocytogenes is more sensitive to D- than to L-lactic acid; however, this difference is less than the strain variation in L-lactic acid sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has implications for the application of lactic acid for food preservation as well as for the understanding of the antibacterial mechanisms of weak organic acids.

Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry.

AIMS: To investigate the antibacterial effect of five essential oils (EO) extracted from aromatic plants (Cymbopogon citratus, Ocimumbasilicum, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale) of Cameroon against strains of Listeria monocytogenes, L. innocua and Staphylococcus aureus. The ability of selected EO to permeabilize the cytoplasmic membrane of L. innocua was also examined. METHODS AND RESULTS: The antibacterial activity of the EO determined by the agar diffusion method showed that T. vulgaris had the highest activity followed by O. gratissimum and C. citratus. Lowest activity was recorded from Z. officinale and O. basilicum. Significant differences in sensitivity between strains of Listeria and S. aureus were observed. Flow cytometry of L. innocua stained with carboxy-fluorescein diacetate showed that the fluorescence intensity of cells exposed to EO decreased faster than nonexposed cells, indicating that EO permeabilized the cytoplasmic membrane with the leakage of carboxy-fluorescein. CONCLUSIONS: Almost all the EO tested showed antibacterial activity to a different extent. The antibacterial effect was due to permeabilization of the cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified the preservative potential of the EO examined. The use of sensitive method, such as flow cytometry, is advantageous for quick generation of data on the antibacterial effect of EO.

Application of Leuconostoc carnosum for biopreservation of cooked meat products.

AIMS: To optimize the practical use of the bacteriocin producing Leuconostoc carnosum 4010 in order to inhibit the growth of Listeria monocytogenes in sliced meat products. METHODS AND RESULTS: Four different methods for biopreservation using the partially purified bacteriocin or the living culture of Leuc. carnosum 4010 were evaluated. The methods using the living protective culture added to the sliced gas packed meat product were more effective in preventing growth of L. monocytogenes than the use of the partially purified leucocins 4010 or bacteriocin produced during fermentation before heat treatment of the saveloy. The application method giving the highest reduction in L. monocytogenes used nozzles for sprinkling the protective culture on all surfaces of each slice of the meat product. In the control samples without the protective culture, L. monocytogenes grew to ca. 107 CFU g(-1), whereas for the application method using nozzles for distributing the protective culture, counts of L. monocytogenes never exceeded 10 CFU g(-1) during 4 weeks of storage at 10 degrees C. CONCLUSIONS: The live cells of the bacteriocin producing Leuc. carnosum 4010 was the most efficient method as it inhibited the growth of L. monocytogenes in cooked, sliced and gas packed saveloy stored at 5 and 10 degrees C for 4 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that biopreservation with lactic acid bacteria is a suitable alternative to chemical preservatives. An even distribution of the protective culture was found to be essential for the efficacy of the protective culture in pilot plant trials.

A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins.

Flow cytometry was investigated as a rapid method to determine the antibacterial effect of the bacteriocins nisin, pediocin PA-1, and sakacin A on the indicator organisms Lactobacillus reuteri DSM 12246, Lactobacillus sakei NCFB 2714 and Lactobacillus sakei DSM 20017, respectively. Fluorescence intensities of the cells were measured by flow cytometry upon exposure to bacteriocins after staining with carboxyfluorescein diacetate (cFDA) and were compared to the number of colony forming units (CFU). The fluorescence index (FI) of the bacterial populations decreased when exposed to the bacteriocins. For the different bacteriocins the pattern of decreases in FI and colony forming units differed at equal bacteriostatic concentrations. FI was the most sensitive measure of bacteriocin activity, i.e. the decrease in FI was observed at lower bacteriocin concentrations than decrease in CFU. It was demonstrated that the decrease in FI was caused by rapid leakage of carboxyfluorescein from cells exposed to pediocin. Cells showing severe leakage after pediocin treatment could be detected as CFU when transferred to a rich medium. Such a repair was less pronounced for cells exposed to sakacin and very limited for cells exposed to nisin. The influence of temperature and NaCl in combination with pediocin on FI and CFU of Lactobacillus sakei NCFB 2714 was examined at conditions relevant to foods. At all temperatures (5, 10, 20 and 37 degrees C) and NaCl concentrations (0, 2 and 4% w/v) investigated the flow cytometric measurements were significantly more sensitive compared to CFU. Both methods showed that the inhibitory effect of pediocin increased with increasing temperatures and decreased with increasing NaCl concentrations.

The Akt/protein kinase B-dependent anti-apoptotic pathway and the mitogen-activated protein kinase cascade are alternatively activated in human glioblastoma multiforme.

We investigated the activation of two important signal transduction pathways in human glioblastoma cells and found a constitutive phosphorylation of either Akt or mitogen-activated protein kinase (MAPK) under serum free conditions. In all but one cell line Wortmannin-sensitive activation of Akt could be attributed to the loss of functional PTEN protein. All cell lines with Akt activation exhibited only weak phosphorylation of the MAPK signal pathway, whereas those without constitutive Akt activation demonstrated high levels of phosphorylated MAPK under serum free conditions. Our data might indicate the presence of two functional subtypes of glioblastoma multiforme, since Akt and MAPK are involved in cellular survival and proliferation signalling, respectively.

Real-time measurements of the interaction between single cells of Listeria monocytogenes and nisin on a solid surface.

A method to obtain real-time measurements of the interactions between nisin and single cells of Listeria monocytogenes on a solid surface was developed. This method was based on fluorescence ratio-imaging microscopy and measurements of changes in the intracellular pH (pH(i)) of carboxyfluorescein succinimidyl ester-stained cells during exposure to nisin. Immobilized cells were placed in a chamber mounted on a microscope and attached to a high-precision peristaltic pump which allowed rapid changes in the nisin concentration. In the absence of nisin, the pH(i) of L. monocytogenes was almost constant (approximately pH 8.0) and independent of the external pH in the pH range from 5.0 to 9.0. In the presence of nisin, dissipation of the pH gradient (DeltapH) was observed, and this dissipation was both time and nisin concentration dependent. The dissipation of DeltapH resulted in cell death, as determined by the number of CFU. In the model system which we used the immobilized cells were significantly more resistant to nisin than the planktonic cells. The kinetics of DeltapH dissipation for single cells revealed a variable lag phase depending on the nisin concentration, which was followed by a very rapid decrease in pH(i) within 1 to 2 min. The differences in nisin sensitivity between single cells in a L. monocytogenes population were insignificant for cells grown to the stationary phase in a liquid laboratory substrate, but differences were observed for cells grown on an agar medium under similar conditions, which resulted in some cells having increased resistance to nisin.

Multiply subtractive kramers-kronig analysis of optical data.

We describe a new, multiply subtractive Kramers-Kronig (MSKK)method to find the optical constants of a material from a singletransmittance or reflectance spectrum covering a small frequencydomain. The MSKK method incorporates independent measurements ofn and k at one or more reference wave-numbervalues to minimize errors due to extrapolations of the data. Anunexpected connection between the MSKK equations and the interpolationtheory allows us to derive the equations from an interpolationtheorem. We found that the locations of the reference points affectthe accuracy of the values determined for the optical constants andthat the optimal spacing of N reference data points isrelated to the zeros of a suitably transformed Chebychev polynomial oforder N. We discuss our efforts to optimize both the numberand the spacing of these reference points and apply our method to sometest spectra.

Fate and effects of the insecticide Dursban 4E in indoor Elodea-dominated and macrophyte-free freshwater model ecosystems: I. Fate and primary effects of the active ingredient chlorpyrifos.

The fate of the insecticide Dursban 4E (active ingredient chlorpyrifos) and its effect on crustaceans and insects was studied in indoor experimental freshwater ecosystems that intended to mimick drainage ditches. A single dose (simulating aerial drift) was applied to achieve nominal chlorpyrifos concentrations of 5 or 35 micrograms/L. Two experiments were performed, one in which all model ecosystems were dominated by the macrophyte Elodea nuttallii, and one using systems devoid of macrophytes. In macrophyte-dominated systems, Elodea vegetation adsorbed a large proportion of the dose applied and hampered the mixing of the insecticide in the water (at least up till day 8). Only a small proportion became incorporated in the sediment. In open water systems the insecticide was rapidly mixed in the water, and the sediment played a very significant role as sink for chlorpyrifos. In both Elodea-dominated and open water systems 50% of the dose applied had disappeared on day 8 post-treatment. The rate of disappearance of chlorpyrifos was relatively rapid in water and macrophytes, and relatively slow in the sediment. Of the arthropods in the zooplankton Cladocera were more susceptible than Copepoda. Significant effects (p less than or equal to 0.05) on Cladocera occurred relatively late in Elodea-dominated systems (in week 4 post-application) in contrast to open water systems (week 1), which is in accordance with the observed differences in the fate of chlorpyrifos. Daphnia pulex, D. longispina and Simocephalus vetulus recovered in the model ecosystems when chlorpyrifos concentrations were lower than 0.1-0.2 micrograms/L, which is in agreement with results of laboratory protocol tests performed with these cladocerans. Among the macroscopic Arthropoda the apparent order of susceptibility was amphipods greater than insects greater than isopods. The isopod Asellus aquaticus was more sensitive to the application of the insecticide than the closely related species Proasellus coxalis. In treated open water systems the latter even increased significantly in numbers. Cage experiments in the model ecosystems performed with several species of Arthropoda indicate that laboratory protocol tests may give a reasonable prediction of short-term direct effects of chlorpyrifos for the same species inhabiting more complex aquatic systems.