Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities.

Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.

Point Mutations in the Monocarboxylate Transporter SLC16A12 Lead to Juvenile and Age-Related Cataract

Purpose: Previously we reported on a premature termination mutation in SLC16A12 that leads to dominant juvenile cataract and renal glucosuria. To assess the mutation rate and genotype-phenotype correlations of SLC16A12 in juvenile or age-related forms of cataract, we performed a mutation screen in cataract patients. Methods: Clinical data of approximately 660 patients were collected, genomic DNA was isolated and analyzed. Exons 3 to 8 including flanking intron sequences of SLC16A12 were PCR amplified and DNA sequence was determined. Selected mutations were tested by cell culture assays, in silico analysis and RT-PCR. Results: We found sequence alterations at a rate of approximately 1/75 patients. None of them was found in 360 control alleles. Alterations affect splice site and regulatory region but most mutations caused an amino acid substitution. The majority of the coding region mutations maps to trans-membrane domains. One mutation located to the 5'UTR. It affects translational efficiency of SLC16A12. In addition, we identified a cataract-predisposing SNP in the non-coding region that causes allele-specific splicing of the 5'UTR region. Conclusions: Altered translational efficiency of the solute carrier SLC16A12 and its allele-specific splicing strongly support a model of challenged homeostasis to cause various forms of cataract. In addition, the pathogenic property of the here reported sequence alterations is supported by the lack of known sequence variations within the coding region of SLC16A12. Due to the relatively high mutation rate, we suggest to include SLC16A12 in diagnostic cataract screening. Generally, our data recommend the assessment of regulatory sequences for diagnostic purposes.

Glutathione precursor, N-acetyl-cysteine, improves mismatch negativity in schizophrenia patients.

In schizophrenia patients, glutathione dysregulation at the gene, protein and functional levels, leads to N-methyl-D-aspartate (NMDA) receptor hypofunction. These patients also exhibit deficits in auditory sensory processing that manifests as impaired mismatch negativity (MMN), which is an auditory evoked potential (AEP) component related to NMDA receptor function. N-acetyl-cysteine (NAC), a glutathione precursor, was administered to patients to determine whether increased levels of brain glutathione would improve MMN and by extension NMDA function. A randomized, double-blind, cross-over protocol was conducted, entailing the administration of NAC (2 g/day) for 60 days and then placebo for another 60 days (or vice versa). 128-channel AEPs were recorded during a frequency oddball discrimination task at protocol onset, at the point of cross-over, and at the end of the study. At the onset of the protocol, the MMN of patients was significantly impaired compared to sex- and age- matched healthy controls (p=0.003), without any evidence of concomitant P300 component deficits. Treatment with NAC significantly improved MMN generation compared with placebo (p=0.025) without any measurable effects on the P300 component. MMN improvement was observed in the absence of robust changes in assessments of clinical severity, though the latter was observed in a larger and more prolonged clinical study. This pattern suggests that MMN enhancement may precede changes to indices of clinical severity, highlighting the possible utility AEPs as a biomarker of treatment efficacy. The improvement of this functional marker may indicate an important pathway towards new therapeutic strategies that target glutathione dysregulation in schizophrenia.

The Fanconi anemia protein FANCM can promote branch migration of Holliday junctions and replication forks.

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Lateralization of olfactory processing: Differential impact of right and left temporal lobe epilepsies.

Olfactory processes were reported to be lateralized. The purpose of this study was to further explore this phenomenon and investigate the effect of the hemispheric localization of epileptogenic foci on olfactory deficits in patients with temporal lobe epilepsy (TLE). Olfactory functioning was assessed in 61 patients and 60 healthy control (HC) subjects. The patients and HC subjects were asked to rate the intensity, pleasantness, familiarity, and edibility of 12 common odorants and then identify them. Stimulations were delivered monorhinally in the nostril ipsilateral to the epileptogenic focus in TLE and arbitrarily in either the left or the right nostril in the HC subjects. The results demonstrated that regardless of the side of stimulation, patients with TLE had reduced performance in all olfactory tasks compared with the HC subjects. With regard to the side of the epileptogenic focus, patients with left TLE judged odors as less pleasant and had more difficulty with identification than patients with right TLE, underlining a privileged role of the left hemisphere in the emotional and semantic processing of odors. Finally, irrespective of group, a tendency towards a right-nostril advantage for judging odor familiarity was found in agreement with a prominent role of the right hemisphere in odor memory processing.

Emotionally negative stimuli can overcome attentional deficits in patients with visuo-spatial hemineglect.

Left unilateral spatial neglect resulting from right brain damage is characterized by loss of awareness for stimuli in the contralesional side of space, despite intact visual pathways. We examined using fMRI whether patients with neglect are more likely to consciously detect in the neglected hemifield, emotionally negative complex scenes rather than visually similar neutral pictures and if so, what neural mechanisms mediate this effect. Photographs of emotional and neutral scenes taken from the IAPS were presented in a divided visual field paradigm. As expected, the detection rate for emotional stimuli presented in the neglected field was higher than for neutral ones. Successful detection of emotional scenes as opposed to neutral stimuli in the left visual field (LVF) produced activations in the parahippocampal and anterior cingulate areas in the right hemisphere. Detection of emotional stimuli presented in the intact right visual field (RVF) activated a distributed network of structures in the left hemisphere, including anterior and posterior cingulate cortex, insula, as well as visual striate and extrastriate areas. LVF-RVF contrasts for emotional stimuli revealed activations in right and left attention related prefrontal areas whereas RVF-LVF comparison showed activations in the posterior cingulate and extrastriate visual cortex in the left hemisphere. An additional analysis contrasting detected vs. undetected emotional LVF stimuli showed involvement of left anterior cingulate, right frontal and extrastriate areas. We hypothesize that beneficial role of emotion in overcoming neglect is achieved by activation of frontal and limbic lobe networks, which provide a privileged access of emotional stimuli to attention by top-down modulation of processing in the higher-order extrastriate visual areas. Our results point to the importance of top-down regulatory role of the frontal attentional systems, which might enhance visual activations and lead to greater salience of emotional stimuli for perceptual awareness.

Atlas-based segmentation of pathological brain MR images

We propose a method for brain atlas deformation inpresence of large space-occupying tumors, based on an apriori model of lesion growth that assumes radialexpansion of the lesion from its starting point. First,an affine registration brings the atlas and the patientinto global correspondence. Then, the seeding of asynthetic tumor into the brain atlas provides a templatefor the lesion. Finally, the seeded atlas is deformed,combining a method derived from optical flow principlesand a model of lesion growth (MLG). Results show that themethod can be applied to the automatic segmentation ofstructures and substructures in brains with grossdeformation, with important medical applications inneurosurgery, radiosurgery and radiotherapy.

Le point sur le traitement de l'hépatite C chronique [An update on the management of chronic hepatitis C]

Treatment of chronic hepatitis C with pegylated interferon-a and ribavirin is now adapted individually based on the virological response on treatment. This approach should improve the tolerability while maintaining or even improving in some patients the efficacy of antiviral therapy. Several new antiviral drugs are currently being evaluated in advanced clinical trials, with very promising results. These new drugs should greatly broaden treatment options for chronic hepatitis C in the near future.