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Background: Irritable bowel syndrome (IBS), defined according to the Rome IV diagnostic criteria, is a chronic functional gastrointestinal disorder characterized by recurrent abdominal pain related to altered bowel habits. First-line recommended treatments are limited to combining drugs targeting predominant symptoms, particularly pain (antispasmodics), constipation (laxatives), and diarrhea (loperamide), yielding only a limited therapeutic gain. GASTRAP ® DIRECT is a class IIa medical formulation composed of a combination of chitin-glucan and simethicone indicated for the symptomatic treatment of gas-related gastrointestinal disorders by combining different mechanisms of action., Aim: To evaluate the efficacy, tolerability, and safety of 4-week GASTRAP ® DIRECT treatment in patients with IBS., Methods: In this prospective, multicenter, open-label trial, 120 patients with IBS received three sticks of GASTRAP ® DIRECT (1.5 g/d of chitin-glucan and 0.75 mg/d of simethicone) per day for 4 weeks. The primary endpoint was the responder rate, defined as the number of patients whose abdominal pain score decreased by ≥ 30% from baseline to week (W) 4. The analysis was performed using the per-protocol set. Cardinal symptoms, impact of global symptoms on daily life, change in stool consistency, and improvement in defecatory disorders were evaluated., Results: Overall, 100 patients were evaluated. At W4, 67% (95%CI: 57-75) showed improvement in abdominal pain (score: 5.8 ± 2.4 vs 2.9 ± 2.0, P < 0.0001). Similar improvements were observed for bloating [8.0 ± 1.7 vs 4.7 ± 2.9, P < 0.0001; 60% (95%CI: 50-70) responders], abdominal distension [7.2 ± 2.1 vs 4.4 ± 3.1, P < 0.0001; 53% (95%CI: 43-63) responders], and impact of global symptoms on daily life [7.1 ± 2.0 vs 4.6 ± 2.9, P < 0.0001; 54% (95%CI: 44-64) responders]. Stool consistency improved in most patients (90% and 57% for patients with liquid and hard stools, respectively). Overall, 42% of patients with defecatory disorders reported very much/considerable improvements by W2. No severe adverse event occurred, and tolerability was rated "good" or "very good" by 93% of patients., Conclusion: GASTRAP ® DIRECT is safe and well tolerated, alleviating IBS symptoms rapidly in 2 weeks. This open-label study suggests that the combination of chitin-glucan and simethicone could be beneficial in patients with IBS., Competing Interests: Conflict-of-interest statement: Desreumaux P reports receiving personal fees from Abbvie, Abbott, Amgen, Biocodex, Biofortis, Biogen, Biokuris, Ferring, Fresenius, Janssen, Kitozyme, Lesaffre, MSD, Norgine, Pfizer, Sandoz, Shire, Takeda, Tillotts, and UCB outside the submitted work. Dr. Desreumaux has a patent (WO2009103884) issued. Veronique Maquet is a product development manager at Kitozyme. Salvatore Modica is chief operating officer at Biokuris, a spin-off company of Kitozyme. Christel Rousseaux reports other from Biotech Companies, outside the submitted work. The other authors declare no conflicts of interest., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

The inspection of very large or thick structures represents one of the biggest challenges for nondestructive techniques. For such objects, a particularly powerful technique is muography, which makes use of free, natural cosmic-ray muons. Among other applications, this technique has been applied to provide two-dimensional (2D) images of nuclear reactors, pyramids, or volcanos. Recently, 3D algorithms developed for medical applications were adapted to the special case of muon imaging to derive density maps. The main difficulties relate to the size of the object and to the limited number of available projections. Here, we report on the first 3D imaging of a whole nuclear reactor, obtained without any prior information on its structure and using the largest set of muographic projections ever made in this field.

Digital twins offer a unique opportunity to design, test, deploy, monitor, and control real-world robotic processes. In this paper we present a novel, modular digital twinning framework developed for the investigation of safety within collaborative robotic manufacturing processes. The modular architecture supports scalable representations of user-defined cyber-physical environments, and tools for safety analysis and control. This versatile research tool facilitates the creation of mixed environments of Digital Models, Digital Shadows, and Digital Twins, whilst standardising communication and physical system representation across different hardware platforms. The framework is demonstrated as applied to an industrial case-study focused on the safety assurance of a collaborative robotic manufacturing process. We describe the creation of a digital twin scenario, consisting of individual digital twins of entities in the manufacturing case study, and the application of a synthesised safety controller from our wider work. We show how the framework is able to provide adequate evidence to virtually assess safety claims made against the safety controller using a supporting validation module and testing strategy. The implementation, evidence and safety investigation is presented and discussed, raising exciting possibilities for the use of digital twins in robotic safety assurance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Douthwaite, Lesage, Gleirscher, Calinescu, Aitken, Alexander and Law.)

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.

RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates.

The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe., (© 2015. Published by The Company of Biologists Ltd.)

The deletion of the protein phosphatase-1 (PP1) regulator known as Nuclear Inhibitor of PP1 (NIPP1) is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B., (© 2014 The Authors.)

Direct composite resin layering techniques preserve sound tooth structure and improve function and esthetics. However, intraoral placement techniques present challenges involving isolation, contamination, individual patient characteristics, and the predictability of restorative outcomes. Computer-aided design and computer-aided manufacturing (CAD/CAM) restorations enable dentists to better handle these variables and provide durable restorations in an efficient and timely manner; however, milled restorations may appear monochromatic and lack proper esthetic characteristics. For these reasons, an uncomplicated composite resin layering restoration technique can be used to combine the benefits of minimally invasive direct restorations and the ease and precision of indirect CAD/CAM restorations. Because most dentists are familiar with and skilled at composite resin layering, the use of such a technique can provide predictable and highly esthetic results. This article describes the layered composite resin restoration technique., (Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

Aurora B is the catalytic subunit of the chromosomal passenger complex (CPC), which coordinates mitotic processes through phosphorylation of key regulatory proteins. In prometaphase, the CPC is enriched at the centromeres to regulate the spindle checkpoint and kinetochore-microtubule interactions. Centromeric CPC binds to histone H3 that is phosphorylated at T3 (H3T3ph) by Aurora B-stimulated Haspin. PP1/Repo-Man acts antagonistically to Haspin and dephosphorylates H3T3ph at the chromosome arms but is somehow prevented from causing a net dephosphorylation of centromeric H3T3ph during prometaphase. Here, we show that Aurora B phosphorylates Repo-Man at S893, preventing its recruitment by histones. We also identify PP2A as a mitotic interactor of Repo-Man that dephosphorylates S893 and thereby promotes the targeting of Repo-Man to chromosomes and the dephosphorylation of H3T3ph by PP1. Thus, Repo-Man-associated PP1 and PP2A collaborate to oppose the chromosomal targeting of Aurora B. We propose that the reciprocal feedback regulation of Haspin and Repo-Man by Aurora B generates a robust bistable response that culminates in the centromeric targeting of the CPC during prometaphase., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The concept of no- or minimal-preparation veneers is more than 25 years old, yet there is no classification system categorizing the extent of preparation for different veneer treatments. The lack of veneer preparation classifications creates misunderstanding and miscommunication with patients and within the dental profession. Such a system could be indicated in various clinical scenarios and would benefit dentists and patients, providing a guide for conservatively preparing and placing veneers. A classification system is proposed to divide preparation and veneering into reduction--referred to as space requirement, working thickness, or material room--volume of enamel remaining, and percentage of dentin exposed. Using this type of metric provides an accurate measurement system to quantify tooth structure removal, with preferably no reduction, on a case-by-case basis, dissolve uncertainty, and aid with multiple aspects of treatment planning and communication.

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Responsible aesthetics and comprehensive care requires a thorough patient examination and evaluation of risk factors that help to prevent and curtail re-restoration and more aggressive tooth preparation. Managing identified risks that could potentially lead to otherwise piecemeal dental procedures keeps dentists treating comprehensively planned cases, not just teeth. Dentistry that is comprehensive, desired, and evidence-based is a valuable service to patients and a satisfying endeavor for dentists and their teams. By choosing between the sure thing of a quick fix and discussing what the patient wants, dentists can embrace a MI, responsible aesthetic approach to treatment planning, case acceptance, and most importantly, patient oral care.

The spindle checkpoint is a mitotic surveillance mechanism that delays anaphase until all sister chromatids are correctly attached to microtubules from opposite poles. Recent studies reveal that protein kinase Aurora B is a key regulator of spindle checkpoint activation whereas protein phosphatase PP1 antagonizes Aurora B and induces checkpoint silencing. Chromosome biorientation stretches the kinetochores and spatially separates centromeric Aurora B from its kinetochore substrates, comprising several PP1-interacting proteins (PIPs). The ensuing dephosphorylation of these PIPs creates docking sites for the bulk recruitment of PP1 to the kinetochores. We propose that this tension-induced targeting of PP1 triggers checkpoint silencing by the dephosphorylation of kinetochore and checkpoint components, including Aurora B substrates. In addition, PP1 also directly inactivates a kinetochore-associated pool of Aurora B and silences checkpoint signaling by opposing the centromeric targeting of Aurora B., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To achieve the benefits that composite restorations can provide, it is incumbent on dentists to understand the importance of proper finishing and polishing techniques and how to incorporate them appropriately into everyday practice. A smooth surface finish is clinically necessary because the presence of surface irregularities from poor finishing and polishing can lead to staining, plaque retention, gingival irritation, recurrent caries, abrasiveness, wear kinetics, and tactile perception by the patient. However, finishing and polishing procedures for direct composite restorations are technique- and material-sensitive. This article describes the proper composite material placement considerations, as well as finishing and polishing techniques and materials, for providing highly esthetic, long-lasting restorations. By incorporating such protocol into their everyday practices, dentists can increase the long-term esthetic and plaque-resistant predictability of direct composite restorations.

The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many different materials and treatment options are available in esthetic and restorative dentistry. Various newer products, such as pressed ceramics, offer enhanced functionality; however, in thinner dimensions, they lack the inherent esthetic beauty of traditional materials such as feldspathic porcelain. As patient demands for better esthetics have increased in recent years, so too has the need for restorative materials that closely mimic the patient's natural dentition. Initially used for the creation of porcelain dentures, feldspathic porcelain has emerged as the premier esthetic material for custom veneer restorations. In recent years, the use of hand-layered powder/liquid feldspathic porcelain has been revived based on its highly esthetic values and little-to-no preparation requirements. By keeping preparation to a minimum, less tooth structure is lost and procedures are much less invasive, which is exactly what patients desire.

Drosophila neuroblasts and mouse radial glial cells can divide asymmetrically to self-renew while producing differentiating daughter cells that contribute to brain growth. Intense research activity in the past few years has started to unveil some of the processes that govern asymmetric division in these two cell types. Here we discuss the case of centrosome asymmetry and the contribution of spindle orientation and non-spindle-related centrosome functions. Although still fragmentary, the emerging picture suggests that both notable parallelisms and striking differences apply.

The concept of minimal preparation is more than 25 years old. Interest in conservative treatments is being revisited as dentistry embraces thinner ceramic veneers and adhesive bonding agents that keep preparations in enamel. Experience and professional knowledge help determine appropriate treatments based on patients' clinical situations and esthetic demands. This article reviews the veneer modality, its role as a conservative treatment, and the protocol to be implemented to ensure proper treatment planning and material selection.

While the importance of protein kinases for the spatial and temporal control of mitotic events has long been recognized, mitotic phosphatases have only recently come into the limelight. It is now well established that protein phosphatases counteract mitotic kinases, so contributing to the generation of switch-like responses at mitotic stage transitions. In addition, the timely dephosphorylation of mitotic phosphoproteins by tightly regulated phosphatases is required for the assembly and stability of the mitotic spindle, the initiation of anaphase, and exit from mitosis. Mitotic phosphatases also emerge as effectors of the DNA damage and spindle assembly checkpoints. These new findings show that protein phosphatases regulate every step of mitosis and provide novel insights into the dynamic and versatile nature of mitotic phosphoregulation.

Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains.

The ubiquitous protein Ser/Thr phosphatase-1 (PP1) interacts with dozens of regulatory proteins that are structurally unrelated. However, most of them share a short, degenerate "RVxF"-type docking motif. Using a broad in silico screening based on a stringent definition of the RVxF motif, in combination with a multistep biochemical validation procedure, we have identified 78 novel mammalian PP1 interactors. A global analysis of the validated RVxF-based PP1 interactome not only provided insights into the conserved features of the RVxF motif but also led to the discovery of additional common PP1 binding elements, described as the "SILK" and "MyPhoNE" motifs. In addition to the doubling of the known mammalian PP1 interactome, our data contribute to the design of PP1 interaction networks. Notably, an interaction network linking PP1 interactors discloses a pleiotropic role of PP1 in cell polarity.

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.

Objective: To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1)., Design: Laboratory experiments., Setting: University hospital., Patient(s): Volunteers who are HIV-1 seronegative and seropositive., Intervention(s): Evaluation of the sensitivity of a reverse-transcriptase (RT)-nested polymerase chain reaction (PCR) in HIV-1 RNA-positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1-seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up., Main Outcome Measure(s): Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control., Result(s): The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration-sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of >/=20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter)., Conclusion(s): The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1-seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.

Couples in whom the man is HIV-1-positive may use medically assisted procreation in order to conceive a child without contaminating the female partner. But, before medically assisted procreation, the semen has to be processed to exclude HIV and tested for HIV nucleic acid before and after processing. The performance was evaluated of the technical protocols used to detect and quantify HIV-1 in 11 centers providing medically assisted procreation for couples with HIV-1 infected men by testing panels of seminal plasma and cells containing HIV-1 RNA and/or DNA. The performance of these tests varied due to the different assays used. False positive results were obtained in 14-19% of cases. The sensitivity for RNA detection in seminal plasma was 500-1,000 RNA copies/ml, over 500 RNA copies/10(6) cells in semen cells, and for DNA detection in semen cells 50-500 DNA copies/10(6) cells. The use of silica-based extraction seemed to increase the assay performance, whereas the use of internal controls to detect PCR inhibitor did not. This first quality control highlights the need for technical improvements of the assays to detect and quantify HIV in semen fractions and for regular evaluation of their performance., ((c) 2006 Wiley-Liss, Inc.)

SIPP1 (splicing factor that interacts with PQBP1 and PP1) is a widely expressed protein of 70 kDa that has been implicated in pre-mRNA splicing. It interacts with protein Ser/Thr phosphatase-1 (PP1) and with the polyglutamine-tract-binding protein 1 (PQBP1), which contributes to the pathogenesis of X-linked mental retardation and neurodegenerative diseases caused by polyglutamine tract expansions. We show here that SIPP1 is a nucleocytoplasmic shuttling protein. Under basal circumstances SIPP1 was largely nuclear, but it accumulated in the cytoplasm following UV- or X-radiation. Nuclear import was mediated by two nuclear localization signals. In addition, SIPP1 could be piggy-back transported to the nucleus with its ligand PQBP1. In the nucleus SIPP1 and PQBP1 formed inclusion bodies similar to those detected in polyglutamine diseases. SIPP1 did not function as a nuclear targeting subunit of PP1 but re-localized nuclear PP1 to storage sites for splicing factors. The C-terminal residues of SIPP1, which do not conform to a classic nuclear export signal, were required for its nuclear export via the CMR-1 pathway. Finally, SIPP1 activated pre-mRNA splicing in intact cells, and the extent of splicing activation correlated with the nuclear concentration of SIPP1. We conclude that SIPP1 is a positive regulator of pre-mRNA splicing that is regulated by nucleocytoplasmic shuttling. These findings also have potential implications for a better understanding of the pathogenesis of X-linked mental retardation and polyglutamine-linked neurodegenerative disorders.

Transcription of human immunodeficiency virus (HIV)-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val36 and Phe38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat.

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1alpha, Arg19 for PP1beta and Arg20 for PP1gamma1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1beta and PP1gamma1 harbours an interaction site for one or more nucleolar interactors.

Strontium titanate SrTiO3(100), (110), and (111) single crystals, undoped or donor doped with up to 1 at% La, were isothermally equilibrated at temperatures between 1523 and 1773 K in synthetic air followed by two different methods of Sr tracer deposition: ion implantation of 87Sr and chemical solution deposition of a thin 86SrTiO3 layer. Subsequently, the samples were diffusion annealed under the same conditions as before. The initial and final depth profiles were measured by SIMS. For strong La-doping both tracer deposition methods yield similar Sr diffusion coefficients, whereas for weak doping the tracer seems to be immobile in the case of ion implantation. The Sr diffusivity does not depend on the crystal orientation, but shows strong dependency on the dopant concentration supporting the defect chemical model that under oxidizing conditions the donor is compensated by Sr vacancies. A comparison with literature data on Sr vacancy, Ti, and La diffusion in this system confirms the concept that all cations move via Sr vacancies. Cation diffusion is several orders of magnitude slower than oxygen diffusion.

Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell, K., and Stark, M. J. R. (2002) J. Cell Sci. 115, 195-206). However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif. Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif. Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1). Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.

Teams practising medically assisted reproduction techniques try to avoid viruses as much as possible. Attitudes towards chronic carriers of viruses are rapidly changing, especially for human immunodeficiency virus (HIV) patients. We focus our attention on the legitimacy of systematic screening before assisted reproductive techniques and the need for specialized approaches including an adapted laboratory for viral hazards as well as the need for a multidisciplinary team. Specificities of HIV, hepatitis C virus (HCV), hepatitis B virus (HBV) carriers and the hypothesis of a reduced fertility potential are discussed. Are male HIV carriers a new indication for assisted reproductive techniques in order to prevent virus transmission? It is largely proven that sperm gradient preparation techniques efficiently decrease viral loads and therefore have a protective effect on contamination risk during assisted reproductive techniques. Although a few thousand assisted reproductive technique cycles were performed in the world for this indication without contamination, it is still too early to demonstrate that this technology is fully safe. Two examples of contaminations during insemination are examined. Many questions remain unresolved, such as the lack of standardized techniques for semen preparation or virus detection or the relative merits of intrauterine insemination or ICSI to prevent HIV contamination during assisted reproductive techniques. The authors plead for well-structured, separate programmes of care linked to research objectives.

Today, in developed countries, many HIV-infected people remain in good health thanks to antiviral medication, and a growing number of them want to have children. The benefit of resorting to assisted procreation and the contamination prevention strategies, throughout pregnancy, are summarized as well as the changes in ethical considerations. The balance between the importance of the message of prevention and the benefit for patients of being assisted in their desire for a child, has evolved towards a growing interest for medical intervention in order to avoid the risks of spontaneous conception outside health care structures. We are presenting the medical structure adapted at Erasme hospital and the 38 first requests taken into account by our pluridisciplinary team. This approach, which is coherent from a scientific point of view, respects both the autonomy of people, carrying HIV as well as the essential interest of the child, in being born uninfected, and also has the enormous advantage of allowing access to parenthood without destroying the consistency and coherence of the message of prevention of sexual contamination.

The problems associated with the ventilation of children who have congenital heart disease with left to righ shunts and pulmonary hypertension are more likely to be acquired than due to the congenital malformation. Haemodynamic, angiographic, bronchoscopic and bronchographic studies demonstrated that ventilation problems arose because of compression of the bronchus by the pulmonary artery. The bronchial compression should be treated by surgery to the heart defect without removing the lung. The intervention should be early to obtain maximum benefit and to avoid irreversible damage to the bronchus.