The Effect of the Stress Induced by Hydrogen Peroxide and Corticosterone on Tryptophan Metabolism, Using Human Neuroblastoma Cell Line (SH-SY5Y).

L-tryptophan (L-Trp) is an important amino acid in several physiological mechanisms, being metabolized into two important pathways: the kynurenine and the serotonin (5-HT) pathways. It is important in processes such as mood and stress response, the 5-HT pathway begins with the conversion of L-Trp to 5-hydroxytryptophan (5-HTP), that is metabolized into 5-HT, converted to melatonin or to 5-hydroxyindoleacetic acid (5-HIAA). Disturbances in this pathway are reported to be connected with oxidative stress and glucocorticoid-induced stress, are important to explore. Thus, our study aimed to understand the role of hydrogen peroxide (H2O2) and corticosterone (CORT)-induced stress on the serotonergic pathway of L-Trp metabolism, and on SH-SY5Y cells, focusing on the study of L-Trp, 5-HTP, 5-HT, and 5-HIAA in combination with H2O2 or CORT. We evaluated the effect of these combinations on cellular viability, morphology, and on the extracellular levels of the metabolites. The data obtained highlighted the different ways that stress induction led to different extracellular medium concentration of the studied metabolites. These distinct chemical transformations did not lead to differences in cell morphology/viability. Additionally, serotonin may be the most sensitive metabolite to the exposure to the different stress inducers, being more promissory to study conditions associated with cellular stress.

Serotonin Type 3 Receptor Is Potentially Involved in Cellular Stress Induced by Hydrogen Peroxide.

Depression is a disease with several molecular mechanisms involved, such as problems in the serotonergic pathway. This disease is very complex and prevalent, and thus important to deeply study and aim to overcome high rates of relapse and therapeutic failure. In this study, two cellular lines were used (HT-22 and SH-SY5Y cells) to gain insight about the role of the serotonin type 3 (5-HT3) receptor in cellular stress induced by hydrogen peroxide and/or corticosterone. In research, these compounds are known to mimic the high levels of oxidative stress and dysfunction of the hypothalamus-hypophysis-adrenal axis by the action of glucocorticoids, usually present in depressed individuals. The receptor 5-HT3 is also known to be involved in depression, previously demonstrated in studies that highlight the role of these receptors as promising targets for antidepressant therapy. Indeed, the drugs used in this work (mirtazapine, scopolamine, and lamotrigine) interact with this serotonergic receptor. Thus, by using cell morphology, cell viability (neutral red and MTT), and HPLC assays, this work aimed to understand the role of these drugs in the stress induced by H2O2/corticosterone to HT-22 and SH-SY5Y cell lines. We concluded that the antagonism of the 5-HT3 receptor by these drugs may be important in the attenuation of H2O2-induced oxidative stress to the cells, but not in the corticosterone-induced stress.

Pharmacological and Non-Pharmacological Agents versus Bovine Colostrum Supplementation for the Management of Bone Health Using an Osteoporosis-Induced Rat Model.

Osteoporosis is defined by loss of bone mass and deteriorated bone microarchitecture. The present study compared the effects of available pharmacological and non-pharmacological agents for osteoporosis [alendronate (ALE) and concomitant supplementation of vitamin D (VD) and calcium (Ca)] with the effects of bovine colostrum (BC) supplementation in ovariectomized (OVX) and orchidectomized (ORX) rats. Seven-month-old rats were randomly allocated to: (1) placebo-control, (2) ALE group (7.5 µg/kg of body weight/day/5 times per week), (3) VD/Ca group (VD: 35 µg/kg of body weight/day/5 times per week; Ca: 13 mg/kg of body weight/day/3 times per week), and (4) BC supplementation (OVX: 1.5 g/day/5 times per week; ORX: 2 g/day/5 times per week). Following four months of supplementation, bone microarchitecture, strength and bone markers were evaluated. ALE group demonstrated significantly higher Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC and significantly lower Ct.Pr, Tb.Pr, Tb.Sp, Ct.BMD and Tb.BMD, compared to placebo (p < 0.05). BC presented significantly higher Ct.Pr, Ct.BMD, Tb.Pr, Tb.Sp, and Tb.BMD and significantly lower Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC compared to ALE in OVX rats (p < 0.05). OVX rats receiving BC experienced a significant increase in serum ALP and OC levels post-supplementation (p < 0.05). BC supplementation may induce positive effects on bone metabolism by stimulating bone formation, but appear not to be as effective as ALE.

Serotonin after ß-Adrenoreceptors' Exposition: New Approaches for Personalized Data in Breast Cancer Cells.

Serotonin is an important monoamine in the human body, playing crucial roles, such as a neurotransmitter in the central nervous system. Previously, our group reported that ß-adrenergic drugs (ICI 118,551, isoprenaline, and propranolol) influence the proliferation of breast cancer cells (MCF-7 cells) and their inherent production of adrenaline. Thus, we aimed to investigate the production of serotonin in MCF-7 cells, clarifying if there is a relationship between this production and the viability of the cells. To address this question, briefly, we treated the MCF-7 cells with ICI 118,551, isoprenaline, and propranolol, and evaluated cellular viability and serotonin production by using MTT, Sulforhodamine B (SRB) and Neutral Red (NR) assays, and HPLC-ECD analysis, respectively. Our results demonstrate that isoprenaline promotes the most pronounced endogenous synthesis of serotonin, about 3.5-fold greater than control cells. Propranolol treatment also increased the synthesis of serotonin (when compared to control). On the other hand, treatment with the drug ICI 118,551 promoted a lower endogenous synthesis of serotonin, about 1.1-fold less than what was observed in the control. Together, these results reveal that MCF-7 cells can produce serotonin, and the drugs propranolol, isoprenaline and ICI 118,551 influence this endogenous production. For the first time, after modulation of the ß-adrenergic system, a pronounced cellular growth can be related to higher consumption of serotonin by the cells, resulting in decreased levels of serotonin in cell media, indicative of the importance of serotonin in the growth of MCF-7 cells.

Bovine Colostrum Supplementation Improves Bone Metabolism in an Osteoporosis-Induced Animal Model.

Osteoporosis is characterized by bone loss. The present study aims to investigate the effects of bovine colostrum (BC) on bone metabolism using ovariectomized (OVX) and orchidectomized (ORX) rat models. Twenty-seven-week-old Wistar Han rats were randomly assigned as: (1) placebo control, (2) BC supplementation dose 1 (BC1: 0.5 g/day/OVX, 1 g/day/ORX), (3) BC supplementation dose 2 (BC2: 1 g/day/OVX, 1.5 g/day/ORX) and (4) BC supplementation dose 3 (BC3: 1.5 g/day/OVX, 2 g/day/ORX). Bone microarchitecture, strength, gene expression of VEGFA, FGF2, RANKL, RANK and OPG, and bone resorption/formation markers were assessed after four months of BC supplementation. Compared to the placebo, OVX rats in the BC1 group exhibited significantly higher cortical bone mineral content and trabecular bone mineral content (p < 0.01), while OVX rats in the BC3 group showed significantly higher trabecular bone mineral content (p < 0.05). ORX rats receiving BC dose 2 demonstrated significantly higher levels of trabecular bone mineral content (p < 0.05). Serum osteocalcin in the ORX was pointedly higher in all BC supplementation groups than the placebo (BC1: p < 0.05; BC2, BC3: p < 0.001). Higher doses of BC induced significantly higher relative mRNA expression of OPG, VEGFA, FGF2 and RANKL (p < 0.05). BC supplementation improves bone metabolism of OVX and ORX rats, which might be associated with the activation of the VEGFA, FGF2 and RANKL/RANK/OPG pathways.

A proposed Common Training Framework for Specialists in Laboratory Medicine under EU Directive 2013/55/EC (The Recognition of Professional Qualifications).

European Union (EU) Directive 2013/55/EC (The Recognition of Professional Qualifications) allows Member States to decide on a common set of minimum knowledge, skills and competences that are needed to pursue a given profession through a Common Training Framework. To be adopted the framework must combine the knowledge, skills and competences of at least one third of the Member States. Professionals who have gained their qualifications under a Common Training Framework will be able to have these recognised automatically within the Union. The backbone of the European Federation of Clinical Chemistry and Laboratory Medicine's (EFLM) proposed Common Training Framework for non-medical Specialists in Laboratory Medicine is outlined here. It is based on an Equivalence of Standards in education, training, qualifications, knowledge, skills, competences and the professional conduct associated with specialist practice. In proposing the recognition of specialist practice EFLM has identified 15 EU Member States able to meet Equivalence and in whom the profession and/or its training is regulated (an additional EU Commission requirement). The framework supports and contributes to the Directive's enabling goals for increasing professional mobility, safeguarding consumers and ensuring a more equitable distribution of skills and expertise across the Member States. It represents EFLM's position statement and provides a template for professional societies and/or competent authorities to engage with the EU Commission.

ß-Adrenoceptor Activation in Breast MCF-10A Cells Induces a Pattern of Catecholamine Production Similar to that of Tumorigenic MCF-7 Cells.

Adrenaline, which participates in the neuroendocrine response that occurs during stress and perimenopause, may be tumorigenic. This exploratory study aimed at investigating whether non-tumorigenic and tumorigenic human breast epithelial cell lines are able to synthesize adrenaline. The study was carried out in non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) human breast cell lines. Expression of enzymes involved in adrenaline synthesis was characterized by RT-qPCR, immunocytochemistry and western blot. Catecholamines and analogue compounds were quantified by HPLC-ECD. Functional assessment of the impact of drugs on cells' tumorigenic potential was assessed by determination of cell viability and clonogenic ability. Both MCF-10A and MCF-7 cells produce catecholamines, but the capacity to produce adrenaline is lower in MCF-10A cells. ß-adrenoceptor activation increases the capacity of MCF-10A cells to produce adrenaline and favor both cell viability and colony formation. It is concluded that exposure of human breast epithelial cells to ß-adrenoceptor agonists increases cell proliferation and the capacity to produce adrenaline, creating an autocrine potential to spread these adrenergic effects in a feed-forward loop. It is conceivable that these effects are related to tumorigenesis, bringing a new perspective to understand the claimed anticancer effects of propranolol and the increase in breast cancer incidence caused by stress or during perimenopause.

Carbidopa Alters Tryptophan Metabolism in Breast Cancer and Melanoma Cells Leading to the Formation of Indole-3-Acetonitrile, a Pro-Proliferative Metabolite.

Carbidopa is used for the treatment of Parkinson's disease (PD) as an inhibitor of DOPA decarboxylase, and PD patients taking carbidopa have a lower incidence of various tumors, except for breast cancer and melanoma. Recently, it was shown that carbidopa inhibits tryptophan-2,3-dioxygenase (TDO) and kynureninase enzymes. In the present study, the effect of carbidopa on the viability and metabolic profile of breast cancer MCF-7 and melanoma A375 cells was investigated. Carbidopa was not effective in inhibiting MCF-7 and A375 proliferation. Liquid chromatography and mass spectrometry revealed a new compound, identified as indole-3-acetonitrile (IAN), which promoted a concentration-dependent increase in the viability of both cell lines. The results suggest that treatment with carbidopa may alter tryptophan (Trp) metabolism in breast cancer and melanoma leading to the formation of a pro-proliferative Trp metabolite, which may contribute to its failure in reducing breast cancers and melanoma incidence in PD patients taking carbidopa.

Serum 25-hydroxyvitamin D levels in multiple sclerosis patients from the north of Portugal.

Increasing evidence has shown that individuals with Multiple Sclerosis (MS) have lower 25-hydroxyvitamin D [25(OH)D] levels compared to healthy controls. There is no information regarding 25(OH)D levels and MS in Portugal. Therefore the aim of the current study was to examine the levels of 25(OH)D in a group of patients with MS and in healthy matched controls, as well as the association of 25(OH)D levels with disease course, disability and severity. A group of 244 unrelated Portuguese patients, with a definitive diagnosis of MS, and 198 ethnically matched healthy controls were included in the study. A sub-group of patients with recent disease onset was included. Serum 25(OH)D was measured using an electrochemiluminescence binding assay. The mean serum level of 25(OH)D in patients with MS was 39.9±22.0 nmol/L, which was significantly lower (p<0.0001) than those in healthy controls, 55.4±23.4 nmol/L. There was a negative correlation between 25(OH)D levels and EDSS (r=-0.293, p<0.0001) and MSSS scores (r=-0.293, p<0.0001). In multiple logistic regression analysis adjusted for age, gender, disease form, EDSS, disease duration and MSSS, 25(OH)D levels were independently associated with EDSS (p=0.004) and disease duration (p=0.016), and with MSSS (p=0.001). In accordance with the majority of the literature, low serum 25(OH)D levels were associated with susceptibility and disability in MS patients from Portugal. Lower serum 25(OH)D levels were also found in patients with a recent disease onset, supporting vitamin D levels as a risk factor for MS.

A triple-biomarker approach for the detection of delayed graft function after kidney transplantation using serum creatinine, cystatin C, and malondialdehyde.

INTRODUCTION: Serum creatinine (SCr) alone does not allow for the early diagnosis of delayed graft function (DGF) following kidney transplantation (KTx). OBJECTIVE, DESIGN AND METHODS: The diagnostic utility of urinary neutrophil gelatinase-associated lipocalin (uNGAL), serum leptin, malondialdehyde (MD.A), and cystatin C (CysC) for the early detection of DGF was previously evaluated by our group in a prospective cohort study of 40 consecutive adults undergoing KTx. Because no single biomarker achieved adequate sensitivity or specificity for practical purposes, this study was designed to evaluate the combined use of new markers with SCr. Urine and blood samples were collected 8-to-12h after KTx (day-1). Logistic regression was used to combine the biomarkers, and receiver operating characteristic curves and areas under the curve (AUC-ROC) were generated. RESULTS: Eighteen recipients developed DGF (dialysis requirement during the first post-transplant week). On day-1, the AUC for SCr to predict DGF was 0.73, 0.88 for uNGAL, 0.90 for MDA, 0.76 for leptin, and 0.91 for CysC. Adding new biomarkers to SCr enhanced the performance of DGF prediction, and the best combination was achieved with SCr, MDA, and CysC (AUC=0.96, sensitivity=100%; specificity=86%). CONCLUSION: A combination of graft damage biomarkers outperformed SCr in the early diagnosis of DGF, and the best performance was achieved by a triple-marker approach, using SCr, MDA, and CysC.

Oxidative stress in kidney transplantation: malondialdehyde is an early predictive marker of graft dysfunction.

BACKGROUND: Oxidative stress is one of the most important components of the ischemia-reperfusion process after kidney transplantation (KTx) and increases with graft dysfunction. METHODS: This prospective study was conducted on 40 consecutive KTx recipients to evaluate time-dependent changes in oxidative stress-related parameters within the first week after KTx and to assess their performance in predicting delayed graft function (DGF=dialysis requirement during initial posttransplant week) and graft function at 1 year. Blood samples were collected before (day 0) and after KTx (days 1, 2, 4, and 7). Total antioxidant capacity, plasma levels of malondialdehyde (MDA), and activities of glutathione peroxidase, glutathione reductase and superoxide dismutase were measured. Multivariable linear mixed and linear regression models, receiver-operating characteristic (ROC), and areas under ROC curves (AUC-ROC) were used. RESULTS: At all time points after KTx, mean MDA levels were significantly higher in patients developing DGF (n=18). Shortly after KTx (8-12 hr), MDA values were higher in DGF recipients (on average, +0.16 µmol/L) and increased further on following day, contrasting with prompt functioning recipients. Day 1 MDA levels accurately predicted DGF (AUC-ROC=0.90), with a performance higher than SCr (AUC-ROC=0.73) and similar to cystatin C (AUC-ROC=0.91). Multivariable analysis revealed that MDA levels on day 7 represented an independent predictor of 1-year graft function. Antioxidant enzyme activities were not significantly changed during the study period and were not predictors of 1-year graft function. CONCLUSIONS: Increased MDA levels on day 1 after KTx might be an early prognostic indicator of DGF, and levels on day 7 might represent a useful predictor of 1-year graft function.

A position paper of the EFLM Committee on Education and Training and Working Group on Distance Education Programmes/E-Learning: developing an e-learning platform for the education of stakeholders in laboratory medicine.

The progress of information and communication technologies has strongly influenced changes in healthcare and laboratory medicine. E-learning, the learning or teaching through electronic means, contributes to the effective knowledge translation in medicine and healthcare, which is an essential element of a modern healthcare system and for the improvement of patient care. E-learning also represents a great vector for the transfer knowledge into laboratory practice, stimulate multidisciplinary interactions, enhance continuing professional development and promote laboratory medicine. The European Federation of Laboratory Medicine (EFLM) has initiated a distance learning program and the development of a collaborative network for e-learning. The EFLM dedicated working group encourages the organization of distance education programs and e-learning courses as well as critically evaluate information from courses, lectures and documents including electronic learning tools. The objectives of the present paper are to provide some specifications for distance learning and be compatible with laboratory medicine practices.

The EC4 European syllabus for post-graduate training in clinical chemistry and laboratory medicine: version 4--2012.

Laboratory medicine's practitioners across the European community include medical, scientific and pharmacy trained specialists whose contributions to health and healthcare is in the application of diagnostic tests for screening and early detection of disease, differential diagnosis, monitoring, management and treatment of patients, and their prognostic assessment. In submitting a revised common syllabus for post-graduate education and training across the 27 member states an expectation is set for harmonised, high quality, safe practice. In this regard an extended 'Core knowledge, skills and competencies' division embracing all laboratory medicine disciplines is described. For the first time the syllabus identifies the competencies required to meet clinical leadership demands for defining, directing and assuring the efficiency and effectiveness of laboratory services as well as expectations in translating knowledge and skills into ability to practice. In a 'Specialist knowledge' division, the expectations from the individual disciplines of Clinical Chemistry/Immunology, Haematology/Blood Transfusion, Microbiology/ Virology, Genetics and In Vitro Fertilisation are described. Beyond providing a common platform of knowledge, skills and competency, the syllabus supports the aims of the European Commission in providing safeguards to increasing professional mobility across European borders at a time when demand for highly qualified professionals is increasing and the labour force is declining. It continues to act as a guide for the formulation of national programmes supplemented by the needs of individual country priorities.

[Iodine intake in Portuguese school children]. / Aporte do Iodo nas Crianças das Escolas em Portugal.

OBJECTIVE: The aim of the present study was to evaluate iodine intake in portuguese school children in order to inform health authorities of eventual measures to be implemented. INTRODUCTION: Iodine is the key element for thyroid hormone synthesis and its deficiency even mild, as found in other European countries, may have deleterious effects in pregnancy resulting in cognitive problems of offsprings. In Portugal there are no recent data on iodine intake in schoolchildren. POPULATION AND METHODS: 3680 children aged 6-12 years of both sexes, from 78 different schools were studied. Iodine intake was evaluated trough urine iodine (UI) determinations using a colorimetic method. RESULTS: The global median UI value was 105.5 µg/L; the percentage of children with UI <100 µg/L was 47.1%, corresponding to 41% of the studied schools. The percentage of values <50 µg/L was 11.8%. The male gender, the south region of the country and the distribution of milk in school were significantly linked with a higher iodine elimination. DISCUSSION: Our global results point to a borderline/ mildly insufficient iodine intake in the portuguese school population. However 47% of the children had UI under 100 µg /L. The comparison of our results with the available data from 30 years ago, point to a considerable improvement, due to silent prophylaxis. Male gender, geographical area and milk distribution influenced positively iodine intake.The importance of milk has been referred in numerous papers. CONCLUSIONS: The study of UI in the Portuguese school population points to a borderline iodine intake. However, in 47% of children iodine intake was inadequate. Compared with data from the eighties, a considerable increase in iodine elimination was found. Taking into account the potencial deleterious effects of inadequate iodine intake, a global prophylaxis with salt iodization has to be considered.

The European Register of Specialists in Clinical Chemistry and Laboratory Medicine: guide to the Register, version 3-2010.

In 1997, the European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) set up a Register for European Specialists in Clinical Chemistry and Laboratory Medicine. The operation of the Register is undertaken by a Register Commission (EC4RC). During the last 12 years, more than 2200 specialists in Clinical Chemistry and Laboratory Medicine have joined the Register. In 2007, EC4 merged with the Forum of European Societies of Clinical Chemistry and Laboratory Medicine (FESCC) to form the European Federation of Clinical Chemistry and Laboratory Medicine (EFCC). Two previous Guides to the Register have been published, one in 1997 and another in 2003. The third version of the Guide is presented in this article and is based on the experience gained and development of the profession since the last revision. Registration is valid for 5 years and the procedure and criteria for re-registration are presented as an Appendix at the end of the article.

The European Register of Specialists in Clinical Chemistry and Laboratory Medicine: Code of Conduct, Version 2--2008.

In 1997, the European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) set up a Register for European Specialists in Clinical Chemistry and Laboratory Medicine. The operation of the Register is undertaken by a Register Commission (EC4RC). During the last 10 years, more than 2000 specialists in Clinical Chemistry and Laboratory Medicine have joined the Register. In 2007, EC4 merged with the Federation of European Societies of Clinical Chemistry and Laboratory Medicine (FESCC) to form the European Federation of Clinical Chemistry and Laboratory Medicine (EFCC). A Code of Conduct was adopted in 2003 and a revised and updated version, taking account particularly of the guidelines of the Conseil Européen des Professions Libérales (CEPLIS) of which EFCC is a member, is presented in this article. The revised version was approved by the EC4 Register Commission and by the EFCC Executive Board in Paris on 6 November, 2008.

EC4 European Syllabus for Post-Graduate Training in Clinical Chemistry and Laboratory Medicine: version 3 - 2005.

The EC4 Syllabus for Postgraduate Training is the basis for the European Register of Specialists in Clinical Chemistry and Laboratory Medicine. The syllabus: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. The syllabus is not primarily meant to be a training guide, but on the basis of the overview given (common minimal programme), national societies should formulate programmes that indicate where knowledge and experience is needed. The main points of this programme are: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. Knowledge in biochemistry, haematology, immunology, etc.; Pre-analytical conditions; Evaluation of results; Interpretations (post-analytical phase); Laboratory management; and Quality insurance management. The aim of this version of the syllabus is to be in accordance with the Directive of Professional Qualifications published on 30 September 2005. To prepare the common platforms planned in this directive, the disciplines are divided into four categories: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. Knowledge in biochemistry, haematology, immunology, etc.; Pre-analytical conditions; Evaluation of results; Interpretations (post-analytical phase); Laboratory management; and Quality insurance management. General chemistry, encompassing biochemistry, endocrinology, chemical (humoral), immunology, toxicology, and therapeutic drug monitoring; Haematology, covering cells, transfusion serology, coagulation, and cellular immunology; Microbiology, involving bacteriology, virology, parasitology, and mycology; Genetics and IVF.

The European Register for Specialists in Clinical Chemistry and Laboratory Medicine: Code of Conduct.

The European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) opened a Register for European Specialists in Clinical Chemistry and Laboratory Medicine in 1997. The operation of the Register is undertaken by a Register Committee (EC4RC). During the last 6 years more than 1500 specialists in clinical chemistry and laboratory medicine have joined the Register. In this article a Code of Conduct for Registrants which was approved at the EC4 Register Committee meeting in Amsterdam, 8 November 2003 is presented.

The European Register for Specialists in Clinical Chemistry and Laboratory Medicine: guide to the Register Version 2-2003 and procedure for re-registration.

The European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) opened a Register for European Chemists in 1997. The operation of the Register is undertaken by a Register Committee (EC4RC). During the last 5 years more than 1,400 clinical chemists entered the register. In this article an update of the first Guide to the Register is given, based on the experience of 5 years of operation and the development of the discipline. The registration is valid for 5 years. In a second part the procedure and the conditions for re-registration are presented.