Optimierung und Vergleich bioinformatischer Methoden zur kalkulierten Karyotypisierung der Akuten Myeloischen Leukämie mittels Next Generation Sequencing

Die akute myeloische Leukämie ist eine genetisch heterogene Erkrankung, die aufgrund von rasantem Verlauf und einer massiven Beeinträchtigung der Betroffenen durch das Verdrängen des Knochenmarks durch die malignen Zellen einer schnellen Diagnosestellung und Therapie bedarf. In aktuellen Klassifikations- und Risikostratifizierungssystemen, die zur Abschätzung der Prognose und damit zur Auswahl einer risikoadaptierten Therapie genutzt werden, stellen molekular- und zytogenetische Charakteristika der Erkrankung die wichtigsten Kriterien dar. Mit next-generation-sequencing-Verfahren ist es möglich, alle relevanten Informationen aus Sequenzierdaten abzuleiten, sofern die Daten mit entsprechend geeigneten bioinformatischen Algorithmen analysiert werden. Im Laufe dieser Arbeit wurde coriandR Software – ChrOmosomal abeRration Identifier AND Reporter in R – ein Tool zur Schätzung des kalkulierten Karyotyps und der copy number variations in den ultra lowcoverage whole-genome sequencing Daten – entwickelt, die in verschiedenen Bereichen der genetisch-onkologischen Diagnostik eingesetzt werden kann bei gleichzeitig geringen Kosten und einer hohen Übereinstimmung mit der Goldstandardmethode der Karyotypisierung – der konventionellen Zytogenetik. Für die Schätzung des kalkulierten Karyotyps und der copy number variations einer Blut- bzw. Tumorprobe wird ein Panel of normals aus den Sequenzierdaten generiert, die aus derselben Gewebeart unter selben Bedingungen aufgearbeitet wurden wie die Blut- bzw. Tumorproben und einen normalen Karyotyp aufweisen (z.B. Blutproben gesunder Probanden oder histologisch tumorfreie Gewebe). Diese Sequenzierungsdaten werden nach dem Alignment durch Bowtie2 mit featureCounts in tabellarische Form überführt und durch die mediane Sequenzierungstiefe pro Bin normalisiert. Im nächsten Schritt erfolgt die Standardisierung mit Bildung der Pseudo-Z-Werte für die Verteilung der Reads in den Bins. Später werden sie mit einer Normalverteilung der Reads in den Bins verglichen. Nach dem Ausschluss von den Bins mit einem abnormen GC-Gehalt und/oder einer großen Varianz kann das Panel of normals für weitere Berechnungen genutzt werden. Die Schätzung des kalkulierten Karyotyps für die Tumorproben basiert auf einem zweiseitigen Normalverteilungstest. Die berechneten p-Werte werden anschließend nach Benjamini-Hochberg-Methode unter Einhaltung der false discovery rate von 5 % adjustiert. Nach dem Erhalt der adjustierten p-Werte werden die abweichenden Bins berechnet. In dem coriandR Bericht werden der Übersichtsplot für die Verteilung der Reads in der Tumorprobe, der kalkulierte Karyotyp, die Liste der copy number variations und die Chromosomenplots abgebildet. Durch die Erstellung eines probenspezifischen Panel of normals kann die Methode von coriandR bei den Sequenzierdaten mit einer unterschiedlichen präanalytischen Aufarbeitung oder mit unterschiedlichen Typen von Ausgangsmaterial (Blut-/Knochenmarkproben oder Proben aus dem Formalin-fixierten Paraffineingebetteten Gewebe) zur Schätzung eines kalkulierten Karyotyps eingesetzt werden. coriandR zeichnet sich durch geringe Laufzeit und die Unabhängigkeit von den Datenformaten anderer Programme aus, so dass die Analyse bereits mit den rohen Sequenzierungsdaten und Eingabe des Geschlechts des Patienten oder der Patientin begonnen werden kann. Die Darstellung der Chromosomenplots mit Giemsa-Banden erlaubt eine einfache Beurteilung durch einen Experten. Die Ergebnisse bei der Schätzung des kalkulierten Karyotyps durch coriandR wurden durch einen Vergleich mit dem Programm Genome Analysis Toolkit validiert und zeigten eine hohe Übereinstimmung mit den Ergebnissen dieses populären Programms. Die kalkulierte Karyotypisierung hat in den Zeiten der sinkenden Kosten für die Genomsequenzierung das Potential, in Kombination mit anderen gezielten Untersuchungen, zum Beispiel Fusionspanels, die konventionelle Zytogenetik bei der Diagnostik der akuten myeloischen Leukämie abzulösen. Darüber hinaus ist es mit dem Ansatz auch möglich, solide Tumoren auf der Ebene von Chromosomensätzen genetisch zu charakterisieren, was wichtige Erkenntnisse für das Verständnis der klinischen Ausprägungen liefern kann

Renal function in children with congenital neurogenic bladder

AIMS: Preservation of renal function in children with congenital neurogenic bladder is an important goal of treatment for the disease. This study analyzed the evolution of renal function in patients with congenital neurogenic bladder. METHODS: We reviewed the records of 58 pediatric patients with respect to the following attributes: gender, age, etiology of neurogenic bladder, reason for referral, medical/surgical management, episodes of treated urinary tract infections, urodynamics, DMSA scintigraphy, weight, height, blood pressure, glomerular filtration rate, microalbuminuria and metabolic acidosis. Statistical analysis was performed, adopting the 5% significance level. RESULTS: The mean age at presentation was 4.2 ± 3.5 years. Myelomeningocele was the most frequent etiology (71.4%). Recurrent urinary tract infection was the reason for referral in 82.8% of the patients. Recurrent urinary tract infections were diagnosed in 84.5% of the patients initially; 83.7% of those patients experienced improvement during follow-up. The initial mean glomerular filtration rate was 146.7 ± 70.1 mL/1.73 m²/min, and the final mean was 193.6 ± 93.6 mL/1.73 m²/min, p = 0.0004. Microalbuminuria was diagnosed in 54.1% of the patients initially and in 69% in the final evaluation. Metabolic acidosis was present in 19% of the patients initially and in 32.8% in the final assessment. CONCLUSIONS: Patient referral to a pediatric nephrologist was late. A reduction in the number of urinary tract infections was observed with adequate treatment, but microalbuminuria and metabolic acidosis occurred frequently despite adequate management

Surfing for Inspiration: digital inspirational material in design practice

Over the last decade, many new opportunities have emerged to support creativity and problem-solving in design by finding inspirational materials via the Internet. Online design communities such as those of Behance and Pinterest showcase portfolios and user-made artwork, and they offer support for designers’ day-to-day work to find and collect inspirational material. However, very little is known about how these communities affect inspiration-related practices of professional designers and how designers view them. This paper presents new data on the practices designers employ when seeking digital inspiration sources online and reflecting on, tracking, and managing them in today’s Web design. Current practice and views on sources of inspiration were described based on responses from 51 professional designers. The results suggest that the Internet has become a prevalent source for ideas in design, yet designers experience mounting issues of trust and relatedness with regard to online sources. Therefore, encouraging both should be considered a guiding principle for tools aimed at supporting designers within the realm of design practice.Peer reviewe

Evaluation of chickenpox susceptibility in children with chronic renal failure

OBJETIVO: Avaliar a suscetibilidade natural à varicela de crianças e adolescentes portadores de insuficiência renal crônica (IRC). MÉTODOS: Estudo transversal de 83 pacientes com idade acima de 18 meses e inferior a 18 anos, durante 2000 e 2001, com ritmo de filtração glomerular (RFG) abaixo de 70mL/min/1,73m², portando cartão vacinal preconizado pela Fundação Nacional de Saúde e que não receberam nenhuma dose da vacina específica. Do total, três pacientes (3/83) foram excluídos, por terem recebido doses da vacina por meio de órgãos não governamentais. A sorologia foi realizada pelo método Enzyme-Linked Immuno Sorbent Assay, considerando-se títulos sorológicos protetores acima de 100mUA/mL. RESULTADOS: Os pacientes renais crônicos tinham idade mediana de 11 anos, 66% eram masculinos, 60% procedentes do próprio município de São Paulo, com RFG médio de 33,6mL/min/1,73m². O diagnóstico clínico de varicela por profissional médico ocorreu em 39 pacientes; destes, 10% se mostraram soronegativos. Dos 80 pacientes restantes, 21 (26%) apresentaram títulos não protetores para varicela. A prevalência de suscetibilidade em menores de seis anos foi 7,93 (IC95%=3,29-19,12) vezes superior à de maiores de seis anos. CONCLUSÕES: Houve diminuição da suscetibilidade à varicela com a idade. Pacientes abaixo de seis anos foram cerca de oito vezes mais suscetíveis à varicela que os renais crônicos com idade mais avançada e duas vezes mais suscetíveis do que a população pediátrica brasileira de mesma idade.OBJECTIVE: To evaluate the immune response to chickenpox natural infection in pediatric patients with chronic renal insufficiency. METHODS: This cross-sectional study enrolled 83 patients between 18 months and 18 years old, with glomerular filtration rate below 70mL/min/1.73m², during the years 2000 and 2001, who did not received specific immunization according to official documentation. Three patients (3/83) had been previously vaccinated by non-governmental agencies and were excluded. Varicella antibodies were evaluated by Enzyme-Linked Immuno Sorbent Assay and antibody titers above 100mUA/mL were considered protective. RESULTS: The median age was 11 years old, 66% were male, 60% lived in the city of São Paulo, with an average glomerular filtration rate of 33.6mL/min/1.73m². Clinical diagnosis of chickenpox was done by pediatricians in 39 patients and four of these 39 were seronegative for varicella. Susceptibility to chickenpox was found in 21/80 (26%) patients and the relative prevalence of susceptibility to the disease was 7.93 (95%CI=3.29-19.12) higher in patients under six years of age. CONCLUSIONS: Susceptibility to varicella infection decreased with age. Patients younger than six years were eight times more susceptible than older patients and twice more susceptible compared to the Brazilian pediatric population of the same age group

Redefining Knee Balance in a Medially Stabilized Prosthesis: An In-Vitro Study

Background To date, there is still no consensus on what soft tissues must be preserved and what structures can be safely released during total knee arthroplasty (TKA) with a medially stabilized implant. Objective The aim of this study was to analyze the effect of a progressive selective release of the medial and lateral soft tissues in a knee implanted with a medially stabilized prosthesis. Method Six cadaveric fresh-frozen full leg specimens were tested. In each case, kinematic pattern and mediolateral laxity were measured in three stages: firstly, prior to implantation; secondly, after the implantation of the trial components, but before any soft tissue release; and thirdly, progressively as soft tissue was released with the trial implant in place. The incremental impact of each selective release on knee balance was then analyzed. Results In all cases sagittal stability was not affected by the progressive release of the lateral soft tissue envelope. It was possible to perform progressive lateral release provided the anterior one-third of the iliotibial band (ITB) remained intact. Progressive medial release could be performed on the medial side provided the anterior fibers of the superficial medial collateral ligament (sMCL) remained intact. Conclusion The medially conforming implant remains stable provided the anterior fibers of sMCL and the anterior fibers of the ITB remain intact. The implant's sagittal stability is mainly dependent on its medial ball-in-socket design

Clinical aspects of autosomal recessive polycystic kidney disease

INTRODUÇÃO: A Doença Renal Policística Autossômica Recessiva (DRPAR) é uma causa importante de morbidade e mortalidade pediátricas, com um espectro variável de manifestações clínicas. MÉTODOS: A apresentação e evolução clínica de 25 pacientes (Pts) foram analisadas através da revisão de prontuários, aplicando-se os formulários propostos por Guay-Woodford et al. As morbidades associadas à doença foram avaliadas quanto à frequência e à idade de manifestação. RESULTADOS: A idade média de diagnóstico foi de 61,45 meses (0 a 336,5 meses), com distribuição similar entre os sexos (52% dos pts do sexo feminino). Houve histórico familiar da doença em 20% dos casos (5/25), com dois casos de consanguinidade. Na análise inicial, diagnosticou-se hipertensão arterial (HAS) em 56% dos Pts (14/25); doença renal crônica estágio > 2 (DRC > 2) em 24% (6/25); infecções do trato urinário (ITU) em 40% (10/25) e hipertensão portal (HP) em 32% dos casos (8/25). Das ultrassonografias abdominais iniciais, 80% demonstraram rins ecogênicos com cistos grosseiros e 64% detectaram fígado e vias biliares normais. Inibidores da ECA foram utilizados em 36% dos Pts, betabloqueadores em 20%, bloqueadores de canais de cálcio em 28% e diuréticos em 36% dos casos. Na análise final, após um tempo de acompanhamento médio de 152,2 meses (29,8 a 274,9 meses), HAS foi diagnosticada em 76% dos Pts, DRC > 2 em 44%, ITU em 52% e HP em 68%. CONCLUSÃO: As altas morbidade e mortalidade associadas à DRPAR justificam a construção de um banco de dados internacional, visando ao estabelecimento de um tratamento de suporte precoce.INTRODUCTION: Autosomal Recessive Polycystic Kidney Disease (ARPKD) is an important pediatric cause of morbidity and mortality, with a variable clinical spectrum. METHODS: The clinical presentation and evolution of 25 patients (Pts) were analyzed by clinical record review, according to the forms proposed by Guay-Woodford et al. Morbidities associated with the disease were evaluated with respect to their frequencies and age of onset. RESULTS: The median age at the diagnosis was 61.45 months (0 to 336.5 months), with similar gender distribution (52% of the patients were female). A family ARPKD history was found in 20% of the cases (5/25), two of them associated with consanguinity. On arrival, arterial hypertension (SAH) was diagnosed in 56% of the Pts (14/25); chronic kidney disease stage > 2 (CKD > 2) in 24% (6/25); urinary tract infection (UTI) in 40% (10/25); and portal hypertension (PH) in 32% of the cases (8/25). Eighty percent of the initial abdominal ultrasonograms detected echogenic kidneys with gross cysts and 64% demonstrated normal liver and biliary ducts. ACE inhibitors were used in 36% of the analyzed patients, beta-blockers in 20%, calcium channel blockers in 28%, and diuretics in 36% of them. In the final evaluation, after an average follow-up time of 152.2 months (29.8 to 274.9 months), SAH was detected in 76% of the cases, CKD > 2 in 44%, UTI in 52% and PH in 68%. CONCLUSION: The high morbidity and mortality associated with ARPKD justify the assembly of an international database, with the aim of establishing an early therapeutic support

Insulin-like Growth Factor I—Releasing Alginate-Tricalciumphosphate Composites for Bone Regeneration

Purpose: Development and characterization of an in situ-forming, osteoconductive, and growth factor-releasing bone implant. Methods: Injectable in situ-forming scaffolds were prepared from a 2% (m/v) alginate solution, tricalciumphosphate (TCP) granules, and poly(lactide-co-glycolide) microspheres (MS), loaded with the osteoinductive growth factor insulin-like growth factor I (IGF-I). Scaffolds were prepared by mixing the components followed by hydrogel formation through calcium carbonate-induced physical cross-linking of the alginate at slightly acidic pH. Physical-chemical properties and cell biocompatibility using osteoblast-like cells (MG-63 and Saos-2) of these scaffolds were investigated. Results: The addition of TCP to the alginate resulted in reduced swelling and gelation time and an increase in stiffness. Osteoblast-like cells (MG-63 and Saos-2) did not show toxic reactions and adhered circumferentially to the TCP granules surface. The addition of the IGF-I MS resulted in an up to sevenfold increased proliferation rate of MG-63 cells as compared to scaffold preparations without IGF-I MS. The alkaline phosphate (ALP) activity—a parameter for osteblastic activity—increased with increasing amounts of TCP in Saos-2 loaded composite scaffolds. Conclusions: A prototype in situ-hardening composite system for conformal filling of bone defects supporting osteoblastic activity for further clinical testing in relevant fracture models was developed and characterize

Influence of nephrotic state on the infectious profile in childhood idiopathic nephrotic syndrome

Patients with idiopathic nephrotic syndrome present alterations in their cellular and humoral immune reactions that predispose them to the development of infectious processes. PURPOSE: To characterize the infectious processes in patients with idiopathic nephrotic syndrome. PATIENTS AND METHODS: Ninety-two children and adolescents with idiopathic nephrotic syndrome were assessed retrospectively. The types of infection were grouped as follows: upper respiratory tract infections; pneumonia; skin infections; peritonitis; diarrhea; urinary tract infection ; herpes virus; and others. The patients were divided into 2 groups: Group I (steroid-responsive) n = 75, with 4 subgroups-IA (single episode) n = 10, IB (infrequent relapsers) n = 5, IC (frequent relapsers) n = 14, and ID (steroid-dependent) n = 46; and Group II (steroid-resistant) n = 17. The incidence-density of infection among the patients was assessed throughout the follow-up period. Comparisons for each group and subgroup were done during the periods of negative and nephrotic proteinuria. RESULTS: The analysis revealed a greater incidence-density of infections during the period of nephrotic proteinuria in all the groups and subgroups, with the exception of subgroup IA. During the period of nephrotic proteinuria, subgroups IC, ID, and Group II presented a greater incidence-density of infections as compared to subgroup IA. For the period of negative proteinuria, there was no difference in the incidence-density of infections between the groups and subgroups. Upper respiratory tract infections were the most frequent infectious processes. CONCLUSION: The nephrotic condition, whether as part of a course of frequent relapses, steroid dependence, or steroid resistance, conferred greater susceptibility to infection among the patients with idiopathic nephrotic syndrome. The results of this study suggest that the best preventive action against infection in this disease is to control the nephrotic state.OBJETIVO: Caracterizar as infecções, em pacientes com Síndrome Nefrótica Idiopática. PACIENTES E MÉTODOS: Foram analisados, os prontuários de 92 crianças e adolescentes com Síndrome Nefrótica Idiopática . Os tipos de infecções foram agrupados em: Infecções de Vias Aéreas Superiores , Pnemonia, Infecções Cutâneas, Peritonite, Diarréia, Infecção do Trato Urinário, Herpes Vírus e Outros. Os pacientes foram divididos, em dois grupos: Grupo I (córtico-sensíveis)-n=75, com quatro subgrupos; IA (episódio único)-n=10; IB (recidivantes infreqüentes)-n=5; IC (recidivantes freqüentes)-n=14 e ID (córtico-dependentes) n=46; e Grupo II (córtico-resistentes)-n=17. Comparou-se a densidade de incidência de infecções nos períodos com proteinúria negativa e nefrótica. No período com proteinúria nefrótica, comparou-se a densidade de incidência de infecções dos grupos e subgrupos entre si. Da mesma forma, no período com proteinúria negativa. RESULTADO: A análise revelou maior densidade de incidência de infecções, no período com proteinúria nefrótica, em todos os grupos e subgrupos, com exceção do subgrupo IA. No período com proteinúria nefrótica, os subgrupos IC, ID e o grupo II, apresentaram maior densidade de incidência de infecções, quando comparados ao subgrupo IA. No período com proteinúria negativa, não houve diferença na densidade de incidência de infecções entre os grupos e subgrupos. As Infecções de Vias Aéreas Superiores foram os processos infecciosos mais freqüentes. CONCLUSÃO: O estado nefrótico, manifesto através de recidivas freqüentes, dependência ou resistência aos corticosteróides, conferiu ao pacientes com Síndrome Nefrótica Idiopática , maior susceptibilidade à infecções. O resultado deste estudo reforça que a melhor prevenção anti-infecciosa nesta doença é o controle do estado nefrótico

Effects of glucocorticoids on growth and bone mineralization

OBJETIVO: Revisar os mecanismos de ações dos glicocorticoides e sua capacidade de induzir osteoporose e déficits de crescimento. FONTES DOS DADOS: A revisão bibliográfica de artigos científicos foi realizada na base de dados MEDLINE e utilizou as palavras-chave agrupadas nas sintaxes “glicocorticoides”, “mineralização óssea”, “crescimento” e “efeitos colaterais”, nos últimos 10 anos, e das referências destes nos reportamos para as publicações mais antigas, mas com os estudos fundamentais para a compreensão do assunto. SÍNTESE DOS DADOS: Destacam-se ações dos glicocorticoides sobre hormônios e citocinas responsáveis pelo crescimento longitudinal. Os efeitos finais dos glicocorticoides sobre o esqueleto são determinados por ações sistêmicas no metabolismo ósseo e por ações diretas desses esteroides nas células ósseas, levando a mudanças no número e função das mesmas e favorecendo a perda óssea. Discutem-se os mecanismos indutores da recuperação dos canais de crescimento e recuperação da massa óssea após a descontinuação dos glicocorticoides; os métodos diagnósticos do metabolismo e mineralização óssea; assim como medidas terapêuticas e preventivas das alterações óssea induzidas pelos glicocorticoides. CONCLUSÃO: A monitorização de cada paciente é essencial para identificação e potencial reversão dos danos associados ao uso crônico de glicocorticoides.OBJECTIVE: To review the various mechanisms of glucocorticoid action and the ability of these agents to induce osteoporosis and growth deficits. SOURCES: A review of the scientific literature was conducted on the basis of a MEDLINE search using the keywords and descriptors “glucocorticoids,” “bone mineralization,” “growth,” and “side effects” and limited to articles published in the last decade. The references cited by these articles were used to identify relevant older publications, with an emphasis on landmark studies essential to an understanding of the topic. SUMMARY OF THE FINDINGS: Emphasis was placed on the actions of glucocorticoids on the hormones and cytokines that modulate linear growth. The end effects of glucocorticoids on the skeletal system are the result of systemic effects on bone metabolism and of direct actions on bone cells, which alter bone cell counts and predispose to bone loss. The mechanisms underlying catch-up growth and bone mass recovery after discontinuation of glucocorticoid treatment are discussed, followed by a review of diagnostic methods available for assessment of bone metabolism and mineralization and of measures for prevention and management of glucocorticoid-induced bone changes. CONCLUSION: Patient monitoring on a case-by-case basis plays an essential role in detection and, potentially, reversal of the damage associated with chronic glucocorticoid therapy