Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases.

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

Expression of SCCmec cassette chromosome recombinases in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

OBJECTIVES: Methicillin resistance in staphylococci is mediated by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is responsible for vertical and horizontal transfer of methicillin resistance. Horizontal transfer implies first SCCmec excision from the chromosome. Site-specific excision is catalysed by the Ccr recombinases, which are encoded by ccrAB genes located on the cassette. The aim of this study is to determine the promoter activity of ccrAB genes in individual cells of methicillin-resistant Staphylococcus aureus (N315, COL and MW2) and Staphylococcus epidermidis (RP62A). One mutant cured of its SCCmec (N315EX) was also used. Exposure to various stresses was included in the study. METHODS: For each strain, translational promoter-green fluorescent protein (gfp) fusions were used to assess the levels of ccr promoter activity in individual cells. Analyses were performed using epifluorescence microscopy and flow cytometry. RESULTS: ccr promoter activity was observed in only a small percentage of cell populations. This 'bistable' phenotype was strain dependent (GFP was expressed in N315 and RP62A, but not in COL and MW2) and growth dependent (GFP-expressing cells decreased from approximately 3% to 1% between logarithmic and stationary growth phases). The ccr promoter of strain N315 displayed normal promoter activity when expressed in SCCmec-negative N315EX. Likewise, the ccr promoter of strain COL (which was inactive in COL) showed normal N315-like activity when transformed into N315 and N315EX. CONCLUSIONS: SCCmec excision operates through bistability, favouring a small fraction of cells to 'sacrifice' their genomic islands for transfer, while the rest of the population remains intact. Determinants responsible for the activity of the ccr promoter were not located on SCCmec, but were elsewhere on the genome. Thus, the staphylococcal chromosome plays a key role in determining SCCmec stability and transferability.

Chromosomal number aberrations and transformation in adult mouse retinal stem cells in vitro.

PURPOSE: The potential of stem cells (SCs) as a source for cell-based therapy on a wide range of degenerative diseases and damaged tissues such as retinal degeneration has been recognized. Generation of a high number of retinal stem cells (RSCs) in vitro would thus be beneficial for transplantation in the retina. However, as cells in prolonged cultivation may be unstable and thus have a risk of transformation, it is important to assess the stability of these cells. METHODS: Chromosomal aberrations were analyzed in mouse RSC lines isolated from adult and from postnatal day (PN)1 mouse retinas. Moreover, selected cell lines were tested for anchorage-dependent proliferation, and SCs were transplanted into immunocompromised mice to assess the possibility of transformation. RESULTS: Marked aneuploidy occurred in all adult cell lines, albeit to different degrees, and neonatal RSCs were the most stable and displayed a normal karyotype until at least passage 9. Of interest, the level of aneuploidy of adult RSCs did not necessarily correlate with cell transformation. Only the adult RSC lines passaged for longer periods and with a higher dilution ratio underwent transformation. Furthermore, we identified several cell cycle proteins that might support the continuous proliferation and transformation of the cells. CONCLUSIONS: Adult RSCs rapidly accumulated severe chromosomal aberrations during cultivation, which led to cell transformation in some cell lines. The culture condition plays an important role in supporting the selection and growth of transformed cells.

A novel study of copy number variations in Hirschsprung disease using the multiple ligation-dependent probe amplification (MLPA) technique.

BACKGROUND Hirschsprung disease (HSCR) is a congenital malformation of the hindgut produced by a disruption in neural crest cell migration during embryonic development. HSCR has a complex genetic etiology and mutations in several genes, mainly the RET proto-oncogene, have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. METHODS In this study we have aimed to analyze the presence of CNVs in some HSCR genes (RET, EDN3, GDNF and ZFHX1B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. RESULTS Two alterations in the MLPA profiles of RET and EDN3 were detected, but a detailed inspection showed that the decrease in the corresponding dosages were due to point mutations affecting the hybridization probes regions. CONCLUSION Our results indicate that CNVs of the gene coding regions analyzed here are not a common molecular cause of Hirschsprung disease. However, further studies are required to determine the presence of CNVs affecting non-coding regulatory regions, as well as other candidate genes.

Phénotype de la trisomie 9q distale chez un enfant présentant un chromosome surnuméraire remanié [t(X;9)]. [Distal 9q trisomy phenotype in a patient with a supernumerary rearranged chromosome [t(X:9)] (author's transl).]

A child with clinical features associated a trisomy for the distal part of 9q was shown to have the following abnormal chromosome complement : 47,XY,+t)X;9) (Xpter yields Xq24:9q31 yields 9qter), inv 9(p11q13), var 14 (14pQFQ34).

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Vectorial capacity, basic reproduction number, force of infection and all that: formal notation to complete and adjust their classical concepts and equations

A dimensional analysis of the classical equations related to the dynamics of vector-borne infections is presented. It is provided a formal notation to complete the expressions for the Ross' Threshold Theorem, the Macdonald's basic reproduction "rate" and sporozoite "rate", Garret-Jones' vectorial capacity and Dietz-Molineaux-Thomas' force of infection. The analysis was intended to provide a formal notation that complete the classical equations proposed by these authors.

Gene copy number polymorphisms in an arbuscular mycorrhizal fungal population.

Gene copy number polymorphism was studied in a population of the arbuscular mycorrhizal fungus Glomus intraradices by using a quantitative PCR approach on four different genomic regions. Variation in gene copy number was found for a pseudogene and for three ribosomal genes, providing conclusive evidence for a widespread occurrence of macromutational events in the population.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

Plastic and evolutionary responses of cell size and number to larval malnutrition in Drosophila melanogaster.

Both development and evolution under chronic malnutrition lead to reduced adult size in Drosophila. We studied the contribution of changes in size vs. number of epidermal cells to plastic and evolutionary reduction of wing size in response to poor larval food. We used flies from six populations selected for tolerance to larval malnutrition and from six unselected control populations, raised either under standard conditions or under larval malnutrition. In the control populations, phenotypic plasticity of wing size was mediated by both cell size and cell number. In contrast, evolutionary change in wing size, which was only observed as a correlated response expressed on standard food, was mediated entirely by reduction in cell number. Plasticity of cell number had been lost in the selected populations, and cell number did not differ between the sexes despite males having smaller wings. Results of this and other experimental evolution studies are consistent with the hypothesis that alleles which increase body size through prolonged growth affect wing size mostly via cell number, whereas alleles which increase size through higher growth rate do so via cell size.

Emergence of young human genes after a burst of retroposition in primates.

The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past approximately 63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome-derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.

Inference about the number of contributors to a DNA mixture: Comparative analyses of a Bayesian network approach and the maximum allele count method.

In the forensic examination of DNA mixtures, the question of how to set the total number of contributors (N) presents a topic of ongoing interest. Part of the discussion gravitates around issues of bias, in particular when assessments of the number of contributors are not made prior to considering the genotypic configuration of potential donors. Further complication may stem from the observation that, in some cases, there may be numbers of contributors that are incompatible with the set of alleles seen in the profile of a mixed crime stain, given the genotype of a potential contributor. In such situations, procedures that take a single and fixed number contributors as their output can lead to inferential impasses. Assessing the number of contributors within a probabilistic framework can help avoiding such complication. Using elements of decision theory, this paper analyses two strategies for inference on the number of contributors. One procedure is deterministic and focuses on the minimum number of contributors required to 'explain' an observed set of alleles. The other procedure is probabilistic using Bayes' theorem and provides a probability distribution for a set of numbers of contributors, based on the set of observed alleles as well as their respective rates of occurrence. The discussion concentrates on mixed stains of varying quality (i.e., different numbers of loci for which genotyping information is available). A so-called qualitative interpretation is pursued since quantitative information such as peak area and height data are not taken into account. The competing procedures are compared using a standard scoring rule that penalizes the degree of divergence between a given agreed value for N, that is the number of contributors, and the actual value taken by N. Using only modest assumptions and a discussion with reference to a casework example, this paper reports on analyses using simulation techniques and graphical models (i.e., Bayesian networks) to point out that setting the number of contributors to a mixed crime stain in probabilistic terms is, for the conditions assumed in this study, preferable to a decision policy that uses categoric assumptions about N.

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design-based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5-10-year-old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5-10-year-old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.

Automated four-color interphase fluorescence in situ hybridization approach for the simultaneous detection of specific aneuploidies of diagnostic and prognostic significance in high hyperdiploid acute lymphoblastic leukemia.

In high hyperdiploid acute lymphoblastic leukemia (ALL), the concurrence of specific trisomies confers a more favorable outcome than hyperdiploidy alone. Interphase fluorescence in situ hybridization (FISH) complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in nondividing cells. To overcome the limits of manual I-FISH, we developed an automated four-color I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10, and 17. Parameters established for nucleus selection and signal detection were evaluated. Cutoff values were determined. Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 patient samples were compared with those obtained with CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed also by I-FISH, and I-FISH revealed numerous additional abnormal clones in all patients, ranging from < or =1% to 31.6% of cells analyzed. We conclude that four-color automated I-FISH permits the identification of concurrent aneuploidies of potential prognostic significance. Large numbers of cells can be analyzed rapidly. The large number of nuclei scored revealed a high level of chromosome variability both at diagnosis and relapse, the prognostic significance of which is of considerable clinical interest and merits further evaluation.

Prevalence of methylphenidate prescription among school-aged children in a Swiss population: increase in the number of prescriptions in the Swiss Canton of Vaud, from 2002 to 2005, and changes in patient demographics.

OBJECTIVE: Methylphenidate is prescribed for children and adolescents to treat ADHD. As in many Western countries, the increase in methylphenidate consumption is a public concern in Switzerland. The article discusses the authors' assessment of prescription prevalence in 2002 and 2005 for school-aged children in the canton of Vaud. METHOD: Pharmacy prescription information is available from the regional public health authority. Descriptive analyses are conducted on an anonymized database of the years 2002 and 2005. Data for each year are compared to assess trends in methylphenidate prescription prevalence. RESULTS: The findings show an increase from 0.74% to 1.02% in the number of prescriptions for 5- to 14-year-old children, particularly in prescriptions for girls. Data also show important geographical differences in prescription. CONCLUSION: The prevalence of methylphenidate prescription is lower in Switzerland than other Western countries, particularly the United States. However, some aspects of prevalence are similar, including the increase per year, demographics, and geographic characteristics.