Showing papers by "Université libre de Bruxelles published in 1999"

Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis

Abstract: The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Ant algorithms for discrete optimization

Abstract: This article presents an overview of recent work on ant algorithms, that is, algorithms for discrete optimization that took inspiration from the observation of ant colonies' foraging behavior, and introduces the ant colony optimization (ACO) metaheuristic. In the first part of the article the basic biological findings on real ants are reviewed and their artificial counterparts as well as the ACO metaheuristic are defined. In the second part of the article a number of applications of ACO algorithms to combinatorial optimization and routing in communications networks are described. We conclude with a discussion of related work and of some of the most important aspects of the ACO metaheuristic.

Ant colony optimization: a new meta-heuristic

Abstract: Recently, a number of algorithms inspired by the foraging behavior of ant colonies have been applied to the solution of difficult discrete optimization problems. We put these algorithms in a common framework by defining the Ant Colony Optimization (ACO) meta-heuristic. A couple of paradigmatic examples of applications of these novel meta-heuristic are given, as well as a brief overview of existing applications.

The ant colony optimization meta-heuristic

Abstract: This chapter contains sections titled: Combinatorial Optimization, The ACO Metaheuristic, How Do I Apply ACO?, Other Metaheuristics, Bibliographical Remarks, Things to Remember, Thought and Computer Exercises

Unresponsiveness to Cannabinoids and Reduced Addictive Effects of Opiates in CB1 Receptor Knockout Mice

Abstract: The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced. These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.

CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo.

Abstract: Cells of the dendritic family display some unique properties that confer to them the capacity to sensitize naive T cells in vitro and in vivo. In the mouse, two subclasses of dendritic cells (DCs) have been described that differ by their CD8α expression and their localization in lymphoid organs. The physiologic function of both cell populations remains obscure. Studies conducted in vitro have suggested that CD8α+ DCs could play a role in the regulation of immune responses, whereas conventional CD8α− DCs would be more stimulatory. We report here that both subclasses of DCs efficiently prime antigen-specific T cells in vivo, and direct the development of distinct T helper (Th) populations. Antigen-pulsed CD8α+ and CD8α− DCs are separated after overnight culture in recombinant granulocyte/macrophage colony-stimulating factor and injected into the footpads of syngeneic mice. Administration of CD8α− DCs induces a Th2-type response, whereas injection of CD8α+ DCs leads to Th1 differentiation. We further show that interleukin 12 plays a critical role in Th1 development by CD8α+ DCs. These findings suggest that the nature of the DC that presents the antigen to naive T cells may dictate the class selection of the adaptative immune response.

SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model

Abstract: In this paper we present SFCHECK, a stand-alone software package that features a unified set of procedures for evaluating the structure-factor data obtained from X-ray diffraction experiments and for assessing the agreement of the atomic coordinates with these data. The evaluation is performed completely automatically, and produces a concise PostScript pictorial output similar to that of PROCHECK [Laskowski, MacArthur, Moss & Thornton (1993). J. Appl. Cryst. 26, 283–291], greatly facilitating visual inspection of the results. The required inputs are the structure-factor amplitudes and the atomic coordinates. Having those, the program summarizes relevant information on the deposited structure factors and evaluates their quality using criteria such as data completeness, structure-factor uncertainty and the optical resolution computed from the Patterson origin peak. The dependence of various parameters on the nominal resolution (d spacing) is also given. To evaluate the global agreement of the atomic model with the experimental data, the program recomputes the R factor, the correlation coefficient between observed and calculated structure-factor amplitudes and Rfree (when appropriate). In addition, it gives several estimates of the average error in the atomic coordinates. The local agreement between the model and the electron-density map is evaluated on a per-residue basis, considering separately the macromolecule backbone and side-chain atoms, as well as solvent atoms and heterogroups. Among the criteria are the normalized average atomic displacement, the local density correlation coefficient and the polymer chain connectivity. The possibility of computing these criteria using the omit-map procedure is also provided. The described software should be a valuable tool in monitoring the refinement procedure and in assessing structures deposited in databases.

Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1

Abstract: Thirty-nine tumor-bearing patients with metastatic melanoma were treated with 3 subcutaneous injections of the MAGE-3.A1 peptide at monthly intervals. No significant toxicity was observed. Of the 25 patients who received the complete treatment, 7 displayed significant tumor regressions. All but one of these regressions involved cutaneous metastases. Three regressions were complete and 2 of these led to a disease-free state, which persisted for more than 2 years after the beginning of treatment. No evidence for a cytolytic T lymphocyte (CTL) response was found in the blood of the 4 patients who were analyzed, including 2 who displayed complete tumor regression. Our results suggest that injection of the MAGE-3.A1 peptide induced tumor regression in a significant number of the patients, even though no massive CTL response was produced.

The use of maximum SOFA score to quantify organ dysfunction/failure in intensive care. Results of a prospective, multicentre study

Abstract: Objective: To evaluate the performance of total maximum sequential organ failure assessment (SOFA) score and a derived measure, delta SOFA (total maximum SOFA score minus admission total SOFA) as a descriptor of multiple organ dysfunction/failure in intensive care. Design: Prospective, multicentre and multinational study. Setting: Forty intensive care units (ICUs) from Australia, Europe, North and South America. Patients: Data on 1,449 patients, evaluated at admission and then consecutively every 24 h until ICU discharge (11,417 records) during May 1995. Excluded from data collection were all patients with a length of stay in the ICU less than 2 days following uncomplicated scheduled surgery. Main outcome measure: Survival status at ICU discharge. Interventions: The collection of raw data necessary for the computation of a SOFA score on admission and then every 24 h, and basic demographic and clinical statistics. Measurements and main results: Mean total maximum SOFA score presented a very good correlation to ICU outcome, with mortality rates ranging from 3.2 % in patients without organ failure to 91.3 % in patients with failure of all the six organs analysed. A maximum score was reached 1.1 ± 0.2 days after admission for all the organ systems analysed. The total maximum SOFA score presented an area under the ROC curve of 0.847 (SE 0.012), which was significantly higher than any of its individual components. The cardiovascular score (odds ratio 1.68) was associated with the highest relative contribution to outcome. No independent contribution could be demonstrated for the hepatic score. No significant interactions were found. Principal components analysis demonstrated the existence of a two-factor structure that became clearer when analysis was limited to the presence or absence of organ failure (SOFA score ≥ 3 points) during the ICU stay. The first factor comprises respiratory, cardiovascular and neurological systems and the second coagulation, hepatic and renal systems. Delta SOFA also presented a good correlation to outcome. The area under the receiver operating characteristic (ROC) curve was 0.742 (SE 0.017) for delta SOFA, lower than the total maximum SOFA score or admission total SOFA score. The impact of delta SOFA on prognosis remained significant after correction for admission total SOFA. Conclusions: The results show that total maximum SOFA score and delta SOFA can be used to quantify the degree of dysfunction/failure already present on ICU admission, the degree of dysfunction/failure that appears during the ICU stay and the cumulative insult suffered by the patient. These properties make it a good instrument to be used in the evaluation of organ dysfunction/failure.

Ant colonies for the quadratic assignment problem

Abstract: This paper presents HAS–QAP, a hybrid ant colony system coupled with a local search, applied to the quadratic assignment problem. HAS–QAP uses pheromone trail information to perform modifications on QAP solutions, unlike more traditional ant systems that use pheromone trail information to construct complete solutions. HAS–QAP is analysed and compared with some of the best heuristics available for the QAP: two versions of tabu search, namely, robust and reactive tabu search, hybrid genetic algorithm, and a simulated annealing method. Experimental results show that HAS–QAP and the hybrid genetic algorithm perform best on real world, irregular and structured problems due to their ability to find the structure of good solutions, while HAS–QAP performance is less competitive on random, regular and unstructured problems.

The Economics of Career Concerns, Part II: Application to Missions and Accountability of Government Agencies

Abstract: The paper uses a simple multitask career concern model in order to analyse the incentives of government agencies' officials. Incentives are impaired by the agency pursuing multiple missions. A lack of focus is even more problematic in the case of fuzzy missions, that is when outsiders are uncertain about the exact nature of the missions actually pursued by the agency. Consequently agencies pursuing multiple missions receive less autonomy. The paper further shows that professionalization creates a sense of mission for the agency, and that the specialization of officials raises their incentives. Last, the paper compares its predictions with the stylized facts on Government bureaucracies. © 1999 The Review of Economic Studies Limited.

The economics of career concerns, part I: Comparing information structures

Abstract: Many incentives in organizations arise not through explicit formal incentive contracts but rather implicitly through career concerns. This paper models career concerns through agents trying to manipulate the market assessment of their future productivity. The information flow from current actions to market assessment is therefore crucial in determining the nature of these incentives. Improved information may either increase or reduce incentives. The impact of information provides a major distinction between the explicit and implicit incentives model. The paper derives general results on comparisons of information structures which serve as counterparts to the standard results on information structures in the principal-agent model: sufficient statistic, impact of a Blackwell garbling, comparison of inclusive information structures. © 1999 The Review of Economic Studies Limited.

The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes.

Abstract: Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-A pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

HIV-1 tat transcriptional activity is regulated by acetylation.

Abstract: The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR). We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR. Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA.

Cabergoline in the treatment of hyperprolactinemia: a study in 455 patients.

Abstract: Cabergoline is a new long-acting dopamine agonist that is very effective and well tolerated in patients with pathological hyperprolactinemia. The aim of this study was to examine, in a very large number of hyperprolactinemic patients, the ability to normalize PRL levels with cabergoline, to determine the effective dose and tolerance, and to assess the effect on clinical symptoms, tumor shrinkage, and visual field abnormalities. We also evaluated the effects of cabergoline in a large subgroup of patients with bromocriptine intolerance or -resistance. We retrospectively reviewed the files of 455 patients (102 males and 353 females) with pathological hyperprolactinemia treated with cabergoline in 9 Belgian centers. Among these patients, 41% had a microadenoma; 42%, a macroadenoma; 16%, idiopathic hyperprolactinemia; and 1%, an empty sella. The median pretreatment serum PRL level was 124 microg/L (range, 16-26,250 microg/L). A subgroup of 292 patients had previously been treated with bromocriptine, of which 140 showed bromocriptine intolerance and 58 showed bromocriptine resistance. Treatment with cabergoline normalized serum PRL levels in 86% of all patients: in 92% of 244 patients with idiopathic hyperprolactinemia or a microprolactinoma and in 77% of 181 macroadenomas. Pretreatment visual field abnormalities normalized in 70% of patients, and tumor shrinkage was seen in 67% of cases. Side effects were noted in 13% of patients, but only 3.9% discontinued therapy because of side effects. The median dose of cabergoline at the start of therapy was 1.0 mg/week but could be reduced to 0.5 mg/week once control was achieved. Patients with a macroprolactinoma needed a higher median cabergoline dose, compared with those with idiopathic hyperprolactinemia or a microprolactinoma: 1.0 mg/week vs. 0.5 mg/week, although a large overlap existed between these groups. Twenty-seven women treated with cabergoline became pregnant, and 25 delivered a healthy child. One patient had an intended abortion and another a miscarriage. In the patients with bromocriptine intolerance, normalization of PRL was reached in 84% of cases, whereas in the bromocriptine-resistant patients, PRL could be normalized in 70%. We confirmed, in a large-scale retrospective study, the high efficacy and tolerability of cabergoline in the treatment of pathological hyperprolactinemia, leaving few patients with unacceptable side effects or inadequate clinical response. Patients with idiopathic hyperprolactinemia or a microprolactinoma, on average, needed only half the dose of cabergoline as those with macroprolactinomas and have a higher chance of obtaining PRL normalization. Cabergoline also normalized PRL in the majority of patients with known bromocriptine intolerance or -resistance. Once PRL secretion was adequately controlled, the dose of cabergoline could often be significantly decreased, which further reduced costs of therapy.

Isolation by distance in a continuous population: reconciliation between spatial autocorrelation analysis and population genetics models

Abstract: Analysis of the spatial genetic structure within continuous populations in their natural habitat can reveal acting evolutionary processes. Spatial autocorrelation statistics are often used for this purpose, but their relationships with population genetics models have not been thoroughly established. Moreover, it has been argued that the dependency of these statistics on variation in mutation rates among loci strongly limits their interest for inferential purposes. In the context of an isolation by distance process, we describe relationships between a descriptor of the spatial genetic structure used in empirical studies, Moran's I statistic and population genetics parameters. In particular, we point out that, when Moran's I statistic is used to describe correlation in allele frequencies at the individual level, it provides an estimator of Wright's coefficient of relationship. We also show that the latter parameter, as a descriptor of genetic structure, is not influenced by selfing rate or ploidy level. Under specific finite population models, numerical simulations show that values of Moran's I statistic can be predicted from analytical theory. These simulations are also used to estimate the time taken to approach a structure at equilibrium. Finally, we discuss the conditions under which spatial autocorrelation statistics are little influenced by variation in mutation rates, so that they could be used to estimate gene dispersal parameters.

Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence.

Abstract: A digital holographic technique is implemented in a microscope for three-dimensional imaging reconstruction. The setup is a Mach–Zehnder interferometer that uses an incoherent light source to remove the coherent noise that is inherent in the laser sources. A phase-stepping technique determines the optical phase in the image plane of the microscope. Out-of-focus planes are refocused by digital holographic computations, thus considerably enlarging the depth of investigation without the need to change the optical focus mechanically. The technique can be implemented in transmission for various magnification ratios and can cover a wide range of applications. Performances and limitations of the microscope are theoretically evaluated. Experimental results for a test target are given, and examples of two applications in particle localization and investigation of biological sample are provided.

Oral versus Intravenous Empirical Antimicrobial Therapy for Fever in Patients with Granulocytopenia Who Are Receiving Cancer Chemotherapy

Abstract: Background Intravenously administered antimicrobial agents have been the standard choice for the empirical management of fever in patients with cancer and granulocytopenia. If orally administered empirical therapy is as effective as intravenous therapy, it would offer advantages such as improved quality of life and lower cost. Methods In a prospective, open-label, multicenter trial, we randomly assigned febrile patients with cancer who had granulocytopenia that was expected to resolve within 10 days to receive empirical therapy with either oral ciprofloxacin (750 mg twice daily) plus amoxicillin–clavulanate (625 mg three times daily) or standard daily doses of intravenous ceftriaxone plus amikacin. All patients were hospitalized until their fever resolved. The primary objective of the study was to determine whether there was equivalence between the regimens, defined as an absolute difference in the rates of success of 10 percent or less. Results Equivalence was demonstrated at the second interim analysis,...

Robust principal component analysis for functional data

Abstract: A method for exploring the structure of populations of complex objects, such as images, is considered. The objects are summarized by feature vectors. The statistical backbone is Principal Component Analysis in the space of feature vectors. Visual insights come from representing the results in the original data space. In an ophthalmological example, endemic outliers motivate the development of a bounded influence approach to PCA.

Dispersion and pollution of the FEM solution for the Helmholtz equation in one, two and three dimensions

Abstract: For high wave numbers, the Helmholtz equation suffers the so-called 'pollution effect'. This effect is directly related to the dispersion. A method to measure the dispersion on any numerical method related to the classical Galerkin FEM is presented. This method does not require to compute the numerical solution of the problem and is extremely fast. Numerical results on the classical Galerkin FEM (p-method) is compared to modified methods presented in the literature. A study of the influence of the topology triangles is also carried out. The efficiency of the different methods is compared. The numerical results in two of the mesh and for square elements show that the high order elements control the dispersion well. The most effective modified method is the QSFEM [1, 2] but it is also very complicated in the general setting. The residual-free bubble [3, 4] is effective in one dimension but not in higher dimensions. The least-square method [1, 5] approach lowers the dispersion but relatively little. The results for triangular meshes show that the best topology is the 'criss-cross' pattern. Copyright © 1999 John Wiley & Sons, Ltd.

Structural Features of Protein−Nucleic Acid Recognition Sites†

Abstract: We analyzed the atomic models of 75 X-ray structures of protein−nucleic acid complexes with the aim of uncovering common properties. The interface area measured the extent of contact between the protein and nucleic acid. It was found to vary between 1120 and 5800 A2. Despite this wide variation, the interfaces in complexes of transcription factors with double-stranded DNA could be broken up into recognition modules where 12 ± 3 nucleotides on the DNA side contact 24 ± 6 amino acids on the protein side, with interface areas in the range 1600 ± 400 A2. For enzymes acting on DNA, the recognition module is on average 600 A2 larger, due to the requirement of making an active site. As judged by its chemical and amino acid composition, the average protein surface in contact with the DNA is more polar than the solvent accessible surface or the typical protein−protein interface. The protein side is rich in positively charged groups from lysine and arginine side chains; on the DNA side the negative charges from pho...

Prenatal stress alters circadian activity of hypothalamo–pituitary–adrenal axis and hippocampal corticosteroid receptors in adult rats of both gender

Abstract: Prenatal stress impairs activity of the hypothalamo-pituitary-adrenal (HPA) axis in response to stress in adult offspring. So far, very few data are available on the effects of prenatal stress on circadian functioning of the HPA axis. Here, we studied the effects of prenatal stress on the circadian rhythm of corticosterone secretion in male and female adult rats. To evaluate the effects of prenatal stress on various regulatory components of corticosterone secretion, we also assessed the diurnal fluctuation of adrenocorticotropin, total and free corticosterone levels, and hippocampal corticosteroid receptors. Finally, in the search of possible maternal factors, we studied the effects of repeated restraint stress on the pattern of corticosterone secretion in pregnant female rats. Results demonstrate that prenatal stress induced higher levels of total and free corticosterone secretion at the end of the light period in both males and females, and hypercorticism over the entire diurnal cycle in females. No diurnal fluctuation of adrenocorticotropin was observed in any group studied. The effects of prenatal stress on corticosterone secretion could be mediated, at least in part, by a reduction in corticosteroid receptors at specific times of day. Results also show that prepartal stress alters the pattern of corticosterone secretion in pregnant females. Those data indicate that prenatally stressed rats exhibit an altered temporal functioning of the HPA axis, which, taken together with their abnormal response to stress, reinforces the idea of a general homeostatic dysfunction in those animals.

Competition, financial discipline and growth

Abstract: This paper develops a general equilibrium model of technological adoption in an economy populated by 'satisficing' entrepreneurs whose main objective is to minimise innovative effort while keeping the firm alive. In such an economy, product market competition is shown to have a stimulating effect on growth. Indeed, by reducing the amount of slack a manager can afford while keeping his firm alive, competition, combined with the threat of liquidation acts as a disciplinary device which fosters technology adoption and therefore growth. We then investigate how the existence of financial markets affects the importance of this growth-enhancing effect of competition.(This abstract was borrowed from another version of this item.)

Limit Cycle Models for Circadian Rhythms Based on Transcriptional Regulation in Drosophila and Neurospora

Abstract: We examine theoretical models for circadian oscillations based on transcriptional regulation in Drosophila and Neurospora. For Drosophila, the molecular model is based on the negative feedback exerted on the expression of the per and tim genes by the complex formed between the PER and TIM proteins. For Neurospora, similarly, the model relies on the feedback exerted on the expres- sion of the frq gene by its protein product FRQ. In both models, sustained rhyth- mic variations in protein and mRNA levels occur in continuous darkness, in the form of limit cycle oscillations. The effect of light on circadian rhythms is taken into account in the models by considering that it triggers degradation of the TIM protein in Drosophila, and frq transcription in Neurospora. When incorporating the control exerted by light at the molecular level, we show that the models can account for the entrainment of circadian rhythms by light-dark cycles and for the damping of the oscillations in constant light, though such damping occurs more readily in the Drosophila model. The models account for the phase shifts induced by light pulses and allow the construction of phase response curves. These com- pare well with experimental results obtained in Drosophila. The model for Droso- phila shows that when applied at the appropriate phase, light pulses of appropri- ate duration and magnitude can permanently or transiently suppress circadian rhythmicity. We investigate the effects of the magnitude of light-induced changes on oscillatory behavior. Finally, we discuss the common and distinctive features of circadian oscillations in the two organisms.

The 1997 spectroscopic GEISA databank

Abstract: The current version GEISA-97 of the computer-accessible database system GEISA (Gestion et Etude des Informations Spectroscopiques Atmospheriques: Management and Study of Atmospheric Spectroscopic Information) is described. This catalogue contains 1,346,266 entries. These are spectroscopic parameters required to describe adequately the individual spectral lines belonging to 42 molecules (96 isotopic species) and located between 0 and 22,656 cm-1. The featured molecules are of interest in studies of the terrestrial as well as the other planetary atmospheres, especially those of the Giant Planets. GEISA-97 contains also a catalog of absorption cross-sections of molecules such as chlorofluorocarbons which exhibit unresolvable spectra. The modifications and improvements made to the earlier edition (GEISA-92) and the data management software are described. GEISA-97 and the associated management software are accessible from the ARA/LMD (Laboratoire de Meteorologie Dynamique du CNRS, France) web site: http://ara01.polytechnique.fr/registration.