Fungicidal effect of human lactoferrin against Candida albicans

Human lactoferrin (LF) in its iron-free state (apo LF), killed Candida albicans in a time- and dose-dependent way. The lethal effect was stronger at pH 7.0 than at pH 5.5 and maximum inhibition at neutral pH was achieved in 25 min when the fungal cells were exposed to LF in 0.05 mM KCl at 37 degrees C. Fe(3+)-saturated LF had no fungicidal activity. Apo LF-mediated killing was also temperature-dependent with enhanced inhibition at higher temperatures (37 degrees, 42 degrees C). The presence of 1 mM D-glucose did not affect the candidacidal activity of apo LF but both phosphate and bicarbonate ions at physiological salivary concentrations completely blocked the anti-fungal effect. Therefore it seems unlikely that LF belongs to the major host defence factors against oral candidosis.     

RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.     

Exome sequencing followed by large-scale genotyping suggests a limited role for moderately rare risk factors of strong effect in schizophrenia

Schizophrenia is a severe psychiatric disorder with strong heritability and marked heterogeneity in symptoms, course, and treatment response. There is strong interest in identifying genetic risk factors that can help to elucidate the pathophysiology and that might result in the development of improved treatments. Linkage and genome-wide association studies (GWASs) suggest that the genetic basis of schizophrenia is heterogeneous. However, it remains unclear whether the underlying genetic variants are mostly moderately rare and can be identified by the genotyping of variants observed in sequenced cases in large follow-up cohorts or whether they will typically be much rarer and therefore more effectively identified by gene-based methods that seek to combine candidate variants. Here, we consider 166 persons who have schizophrenia or schizoaffective disorder and who have had either their genomes or their exomes sequenced to high coverage. From these data, we selected 5,155 variants that were further evaluated in an independent cohort of 2,617 cases and 1,800 controls. No single variant showed a study-wide significant association in the initial or follow-up cohorts. However, we identified a number of case-specific variants, some of which might be real risk factors for schizophrenia, and these can be readily interrogated in other data sets. Our results indicate that schizophrenia risk is unlikely to be predominantly influenced by variants just outside the range detectable by GWASs. Rather, multiple rarer genetic variants must contribute substantially to the predisposition to schizophrenia, suggesting that both very large sample sizes and gene-based association tests will be required for securely identifying genetic risk factors.     

Methane-cycling microbial communities and methane emission in natural and restored peatlands

We addressed how restoration of forestry-drained peatlands affects CH(4)-cycling microbes. Despite similar community compositions, the abundance of methanogens and methanotrophs was lower in restored than in natural sites and correlated with CH(4) emission. Poor establishment of methanogens may thus explain low CH(4) emissions on restored peatlands even 10 to 12 years after restoration.     

PSEUDOMARKER: a powerful program for joint linkage and/or linkage disequilibrium analysis on mixtures of singletons and related individuals

A decade ago, there was widespread enthusiasm for the prospects of genome-wide association studies to identify common variants related to common chronic diseases using samples of unrelated individuals from populations. Although technological advancements allow us to query more than a million SNPs across the genome at low cost, a disappointingly small fraction of the genetic portion of common disease etiology has been uncovered. This has led to the hypothesis that less frequent variants might be involved, stimulating a renaissance of the traditional approach of seeking genes using multiplex families from less diverse populations. However, by using the modern genotyping and sequencing technology, we can now look not just at linkage, but jointly at linkage and linkage disequilibrium (LD) in such samples. Software methods that can look simultaneously at linkage and LD in a powerful and robust manner have been lacking. Most algorithms cannot jointly analyze datasets involving families of varying structures in a statistically or computationally efficient manner. We have implemented previously proposed statistical algorithms in a user-friendly software package, PSEUDOMARKER. This paper is an announcement of this software package. We describe the motivation behind the approach, the statistical methods, and software, and we briefly demonstrate PSEUDOMARKER's advantages over other packages by example.     

Lentiviral vector-derived shRNAs confer enhanced suppression of Semliki forest virus replication in BHK-21 cells compared to shRNAs expressed from plasmids

Semliki forest virus (SFV) is a pathogen causing lethal encephalitis in laboratory mice. In this study, we obtained three short hairpin RNAs (shRNAs) which could specifically target SFV sequence in GFP reporting systems and effectively suppress SFV replication in luciferase-containing reporter virus system. At a multiplicity of infection (MOI) of 0.001, the luciferase reporter activity was reduced by 78–92% by shRNA expression plasmids and virus yields reduced 2 to 10-fold at 20 h post-infection. When lentiviral vector-derived shRNAs were employed, the virus titers decreased 8 to 126-fold at 24 h post-infection and 6 to 19-fold at 48 h post-infection and the cell survival was prolonged. These data formed the basis for further in vivo studies of RNA interference in mouse models.     

Regulation of the sequential processing of Semliki Forest virus replicase polyprotein

The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123+nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20-30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1-4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.     

A New Rapid On-Line Imaging Method to Determine Particle Size Distribution of Granules

The purpose of this research was to study the feasibility of the new image analysis method in the particle size determination of the granules. The method is capable of forming a three-dimensional topographic image of a sample surface from a digital picture. In the method, a flat granule bed surface was illuminated from three different directions, using the three primary colors (red, green, and blue). One color picture was taken by a digital camera, after which a topographic image of the object surface was constructed. The particle size distribution was then calculated from the image data. The particle size analysis method was tested both off-line and on-line. Off-line particle size measurement results determined by the image analysis method corresponded quite well to those of sieve analysis in the size fraction range 250–1,000 μm. In on-line application, images were successfully retrieved and median granule size trend could be calculated and followed during fluid bed granulations.