Self-interaction of human Pex11pβ during peroxisomal growth and division.

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright: © 2013 Bonekamp et al.Pex11 proteins are involved in membrane elongation and division processes associated with the multiplication of peroxisomes. Human Pex11pβ has recently been linked to a new disorder affecting peroxisome morphology and dynamics. Here, we have analyzed the exact membrane topology of Pex11pβ. Studies with an epitope-specific antibody and protease protection assays show that Pex11pβ is an integral membrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix and N- and C-termini facing the cytosol. A glycine-rich internal region within Pex11pβ is dispensable for peroxisome membrane elongation and division. However, we demonstrate that an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids is crucial for membrane elongation and self-interaction of Pex11pβ. Interestingly, we find that Pex11pβ self-interaction strongly depends on the detergent used for solubilization. We also show that N-terminal cysteines are not essential for membrane elongation, and that putative N-terminal phosphorylation sites are dispensable for Pex11pβ function. We propose that self-interaction of Pex11pβ regulates its membrane deforming activity in conjunction with membrane lipids.Portuguese Foundation for Science and Technology (FCT)FEDERCRUP/DAA

POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA(+) Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression

Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

This is the author accepted manuscript. The final version is available from Company of Biologists via the DOI in this record.Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the Cterminus, anchoring them to organelle membranes where they mediate a variety of critical cellular processes. Mutations in individual TA proteins cause a number of severe inherited disorders. However, the molecular mechanisms and signals facilitating proper TA protein targeting are not fully understood, in particular in mammals. Here, we identify additional TA proteins at peroxisomes or shared by multiple organelles in mammals and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelles. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We demonstrate that subtle alterations in those physicochemical parameters can shift TA protein targeting between organelles, explaining why peroxisomes and mitochondria share many TA proteins. Our analyses enabled us to allocate characteristic physicochemical parameters to different organelle groups. This classification allows for the first time, successful prediction of the location of uncharacterized TA proteins.We thank colleagues who provided materials (see Tables S1-S4) and acknowledge support from A. C. Magalhães, M. Almeida, D. Tuerker, S. Kuehl and C. Davies. This work was supported by the Biotechnology and Biological Sciences Research Council (BB/K006231/1 to M.S.), a Wellcome Trust Institutional Strategic Support Award (WT097835MF, WT105618MA to M.S.), the Portuguese Foundation for Science and Technology and FEDER/COMPETE (PTDC/BIA-BCM/118605/2010 to M.S.; SFRH/BD/37647/2007 to N.B.; SFRH/BPD/77619/2011 and UID/BIM/04501/2013 to D.R.). M.W., E.A.G., and M.S. are supported by Marie Curie Initial Training Network (ITN) action PerFuMe (316723)

Two randomized crossover multicenter studies investigating gastrointestinal symptoms after bread consumption in individuals with noncoeliac wheat sensitivity: do wheat species and fermentation type matter?

Background: Many individuals reduce their bread intake due to the belief that wheat is the cause of their gastrointestinal (GI) symptoms. Different grains and processing methods may impact tolerability. Objective: We investigated the effects of six different types of bread on GI symptoms in individuals with self-reported non-coeliac wheat sensitivity (NCWS). Methods: Two parallel randomised double-blind crossover multicentre studies were conducted. NCWS individuals, in whom coeliac disease and wheat allergy were ruled out, received five slices of (study A, n=20) yeast fermented (YF) or (study B, n=20) sourdough fermented (SF) bread made of bread wheat, spelt or emmer on three separate intervention days. Each test day was preceded by a run-in period of 3 days and separated by a wash-out period of at least 7 days. GI symptoms were evaluated by change in symptom score (test day minus average of the 3-day run-in period) on a 0-100mm visual analogue scale (delta VAS). Responders were defined as an increase in delta VAS of at least 15mm for overall GI symptoms, abdominal discomfort, abdominal pain, bloating and/or flatulence. Results: The overall change in GI symptoms did not differ between breads of different grains (YF p=0.267; SF p=0.144). The number of responders was also comparable for both YF (6 to wheat, 5 to spelt, and 7 to emmer, p=0.761) and SF breads (9 to wheat, 7 to spelt, and 8 to emmer, p=0.761). Conclusion: The majority of NCWS individuals experienced GI symptoms for at least one of the breads, but on a group level, no differences were found between different grain types for either YF or SF breads. Clinical Trial Registry: ClinicalTrials.gov, NCT0408447

Three-dimensional Magnetic Resonance Imaging–based Printed Models of Prostate Anatomy and Targeted Biopsy-proven Index Tumor to Facilitate Patient-tailored Radical Prostatectomy—A Feasibility Study

In this prospective single-center feasibility study, we demonstrate that the use of three-dimensional (3D)-printed prostate models support nerve-sparing radical prostatectomy (RP) and intraoperative frozen sectioning (IFS) in ten men suffering from intermediate- and high-risk prostate cancer (PC), of whom seven harbored pT3 disease. Patient-specific 3D resin models were printed based on preoperative multiparametric magnetic resonance imaging (mpMRI) to provide an exact 3D impression of significant tumor lesions. RP and IFS were planned in a patient-tailored fashion. The 36-region Prostate Imaging Reporting and Data System (PI-RADS) v2.0 scheme was used to compare the MRI/3D print with whole-mount histopathology. In all cases, localization of the index lesion was correctly displayed by MRI and the 3D model. Localization of significant PC lesions correlated significantly (Pearson`s correlation coefficient of 0.88; p <  0.001). In addition, a significant correlation of the width, length, and volume of the tumor and prostate gland, derived from the printed model and histopathology, was found, using Pearson's correlation analyses and Bland-Altman plots. In conclusion, 3D-printed prostate models correlate well with final pathology and can be used to tailor RP. PATIENT SUMMARY: The use of three-dimensional (3D)-printed prostate models based on preoperative magnetic resonance imaging (MRI) may improve prostatectomy outcome. This study confirmed the accuracy of 3D-printed prostates compared with pathology from radical prostatectomy specimens. Thus, MRI-derived 3D-printed prostate models can assist in prostate cancer surgery

Detection of Significant Prostate Cancer Using Target Saturation in Transperineal Magnetic Resonance Imaging/Transrectal Ultrasonography-fusion Biopsy

BACKGROUND: Multiparametric magnetic resonance imaging (mpMRI) and targeted biopsies (TBs) facilitate accurate detection of significant prostate cancer (sPC). However, it remains unclear how many cores should be applied per target. OBJECTIVE: To assess sPC detection rates of two different target-dependent magnetic resonance imaging (MRI)/transrectal ultrasonography (TRUS)-fusion biopsy approaches (TB and target saturation [TS]) compared with extended systematic biopsies (SBs). DESIGN, SETTING, AND PARTICIPANTS: Retrospective single-centre outcome of transperineal MRI/TRUS-fusion biopsies of 213 men was evaluated. All men underwent TB with a median of four cores per MRI lesion, followed by a median of 24 SBs, performed by experienced urologists. Cancer and sPC (International Society of Urological Pathology grade group ≥2) detection rates were analysed. TB was compared with SB and TS, with nine cores per target, calculated by the Ginsburg scheme and using individual cores of the lesion and its "penumbra". OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cancer detection rates were calculated for TS, TB, and SB at both lesion and patient level. Combination of SB + TB served as a reference. Statistical differences in prostate cancer (PC) detection between groups were calculated using McNemar's tests with confidence intervals. RESULTS AND LIMITATIONS: TS detected 99% of 134 sPC lesions, which was significantly higher than the detection by TB (87%, p = 0.001) and SB (82%, p < 0.001). SB detected significantly more of the 72 low-risk PC lesions than TB (99% vs 68%, p < 0.001) and 10% (p = 0.15) more than that detected by TS. At a per-patient level, 99% of men harbouring sPC were detected by TS. This was significantly higher than that by TB and SB (89%, p = 0.03 and 81%, p = 0.001, respectively). Limitations include limited generalisability, as a transperineal biopsy route was used. CONCLUSIONS: TS detected significantly more cases of sPC than TB and extended SB. Given that both 99% of sPC lesions and men harbouring sPC were identified by TS, the results suggest that this approach allows to omit SB cores without compromising sPC detection. PATIENT SUMMARY: Target saturation of magnetic resonance imaging-suspicious prostate lesions provides excellent cancer detection and finds fewer low-risk tumours than the current gold standard combination of targeted and systematic biopsies

The Sentinel-3 Mission Performance Center

As part of the Sentinel-3 mission and in order to ensure the highest quality of products, ESA in cooperation with EUMETSAT has set up the Sentinel-3 Mission Performance Centre (S-3 MPC). This facility is part of the Payload Data Ground Segment (PDGS) and aims at controlling the quality of all generated products, from L0 to L2. The S-3 MPC is composed of a Coordinating Centre (CC), where the core infrastructure is hosted, which is in charge of the main routine activities (especially the quality control of data) and the overall service management. Expert Support Laboratories (ESLs) are involved in calibration and validation activities and provide specific assessment of the products (e.g., analysis of trends, ad hoc analysis of anomalies, etc.). The S-3 MPC interacts with the Processing Archiving Centres (PACs) and the Marine centre at EUMETSAT