Lateralization of lateral displays in convict cichlids

We examine lateralization of lateral displays in convict cichlids, Amatitlania nigrofasciata, and show a population level preference for showing the right side. This enables contesting pairs of fish to align in a head-to-tail posture, facilitating other activities. We found individuals spent a shorter mean time in each left compared with each right lateral display. This lateralization could lead to contesting pairs using a convention to align in a predictable head-to-tail arrangement to facilitate the assessment of fighting ability. It has major implications for the common use of mirror images to study fish aggression, because the 'opponent' would never cooperate and would consistently show the incorrect side when the real fish shows the correct side. With the mirror, the 'normal' head-to-tail orientation cannot be achieved.     

Alzheimer-specific variants in the 3'UTR of Amyloid precursor protein affect microRNA function

Background

APP expression misregulation can cause genetic Alzheimer's disease (AD). Recent evidences support the hypothesis that polymorphisms located in microRNA (miRNA) target sites could influence the risk of developing neurodegenerative disorders such as Parkinson's disease (PD) and frontotemporal dementia. Recently, a number of single nucleotide polymorphisms (SNPs) located in the 3'UTR of APP have been found in AD patients with family history of dementia. Because miRNAs have previously been implicated in APP expression regulation, we set out to determine whether these polymorphisms could affect miRNA function and therefore APP levels.

Results

Bioinformatics analysis identified twelve putative miRNA bindings sites located in or near the APP 3'UTR variants T117C, A454G and A833C. Among those candidates, seven miRNAs, including miR-20a, miR-17, miR-147, miR-655, miR-323-3p, miR-644, and miR-153 could regulate APP expression in vitro and under physiological conditions in cells. Using luciferase-based assays, we could show that the T117C variant inhibited miR-147 binding, whereas the A454G variant increased miR-20a binding, consequently having opposite effects on APP expression.

Conclusions

Taken together, our results provide proof-of-principle that APP 3'UTR polymorphisms could affect AD risk through modulation of APP expression regulation, and set the stage for further association studies in genetic and sporadic AD.     

Performance of ultra-deep pyrosequencing in analysis of HIV-1 pol gene variation

Introduction

Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed.

Principal Finding

In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858–26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11–0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log₁₀ of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads.

Conclusion

This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage.     

Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.     

Female sex and IL28B, a synergism for spontaneous viral clearance in hepatitis C virus (HCV) seroconverters from a community-based cohort

Background & Aims

Since acute hepatitis C virus (HCV) infection is often asymptomatic, it is difficult to examine the rate and determinants of spontaneous clearance. Consequently, these studies are subject to bias, which can potentially lead to biased rates of viral clearance and risk estimates. We evaluated determinants of spontaneous HCV clearance among HCV seroconverters identified in a unique community-based cohort.

Methods

Subjects were 106 drug users with documented dates of HCV seroconversion from the Amsterdam Cohort Study. Logistic regression was used to examine sociodemographic, behavioral, clinical, viral and host determinants, measured around acute infection, of HCV clearance.

Results

The spontaneous viral clearance rate was 33.0% (95% confidence interval (CI) 24.2–42.8). In univariate analyses female sex and fever were significantly associated with spontaneous clearance. The favorable genotypes for rs12979860 (CC) and rs8099917 (TT) were associated with spontaneous clearance, although borderline significant. In multivariate analysis, females with the favorable genotype for rs12979860 (CC) had an increased odds to spontaneously clear HCV infection (adjustedOR 6.62, 95% 2.69–26.13), whereas females with the unfavorable genotype were as likely as men with the favorable and unfavorable genotype to clear HCV. Chronic Hepatitis B infection and absence of HIV coinfection around HCV seroconversion also favor HCV clearance.

Conclusions

This study shows that co-infection with HIV and HBV and genetic variation in the IL28B region play an important role in spontaneous clearance of HCV. Our findings suggest a possible synergistic interaction between female sex and IL28B in spontaneous clearance of HCV.     

Antibodies against alpha-synuclein reduce oligomerization in living cells

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.     

Cryosurgery with pulsed electric fields

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion.     

The Postnatal Development of the Cerebellum— A fMRI and Silver Study

Summary The aim of this study was to evaluate the postnatal development of the cerebella of the pig and to compare this with the activation of the fMRI. The cells in the cerebella were studied by silver technique and the activation of the fMRI in the cerebella was initiated by flexion and extension of the hind paw. Our results showed an increase of the branching of the cells of the cerebellar cortex postnatally, coordinated with registration of fMRI active sites in the cerebella at 6-month postnatal. We concluded that the full maturation of the cerebella was around 6-month postnatal in the pig.