Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients

BACKGROUND: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction. METHODS: The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed. RESULTS: CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84). CONCLUSION: Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined

array CGH screening of 134 unrelated families

Background A growing number of non-coding regulatory mutations are being identified in congenital disease. Very recently also some exons of protein coding genes have been identified to act as tissue specific enhancer elements and were therefore termed exonic enhancers or “eExons”. Methods We screened a cohort of 134 unrelated families with split-hand/split-foot malformation (SHFM) with high resolution array CGH for CNVs with regulatory potential. Results In three families with an autosomal dominant non-syndromic SHFM phenotype we detected microdeletions encompassing the exonic enhancer (eExons) 15 and 17 of DYNC1I1. In a fourth family, who had hearing loss in addition to SHFM, we found a larger deletion of 510 kb including the eExons of DYNC1I1 and, in addition, the human brain enhancer hs1642. Exons 15 and 17 of DYNC1I1 are known to act as tissue specific limb enhancers of DLX5/6, two genes that have been shown to be associated with SHFM in mice. In our cohort of 134 unrelated families with SHFM, deletions of the eExons of DYNC1I1 account for approximately 3% of the cases, while 17p13.3 duplications were identified in 13% of the families, 10q24 duplications in 12%, and TP63 mutations were detected in 4%. Conclusions We reduce the minimal critical region for SHFM1 to 78 kb. Hearing loss, however, appears to be associated with deletions of a more telomeric region encompassing the brain enhancer element hs1642. Thus, SHFM1 as well as hearing loss at the same locus are caused by deletion of regulatory elements. Deletions of the exons with regulatory potential of DYNC1I1 are an example of the emerging role of exonic enhancer elements and their implications in congenital malformation syndromes

Особенности транспортной логистики сборных грузов в сфере ВЭД

Перемещение товаров в составе сборного груза может быть выгодным совершенно разным субъектам. Для крупных компаний это удобно для доставки разнородных товаров и пробных образцов, а для более мелких позволяет оптимизировать оборотные средства. Также огромную часть клиентов транспортных компаний составляют ретейловые компании и индивидуальные предприниматели, объемы поставок которых не позволяют экономически выгодно перемещать свои товары иначе. Движение товара в составе сборного груза имеет ряд неоспоримых преимуществ, такие как: упрощение отслеживания товаров и сокращение транспортных издержек, благодаря которым всё больше компаний и физических лиц обращаются к данному способу перемещения товаров, хоть и время доставки увеличивается из-за дополнительных этапов в перемещении товара.The movement of goods as part of a consolidated cargo can be beneficial to completely different entities. For large companies, it is convenient for the delivery of heterogeneous goods and test samples, and for smaller ones, it allows optimizing working capital. Also, a huge part of the customers of transport companies are retail companies and individual entrepreneurs, the volume of supply of which does not allow economically move their goods otherwise. The movement of goods as part of the consolidated cargo has a number of undeniable advantages, such as: simplification of tracking of goods and reduction of transport costs, thanks to which more and more companies and individuals are turning to this method of movement of goods, although the delivery time is increased due to additional step

Phenotypic overlap in the contribution of individual genes to CNV pathogenicity revealed by cross-species computational analysis of single-gene mutations in humans, mice and zebrafish

SUMMARY Numerous disease syndromes are associated with regions of copy number variation (CNV) in the human genome and, in most cases, the pathogenicity of the CNV is thought to be related to altered dosage of the genes contained within the affected segment. However, establishing the contribution of individual genes to the overall pathogenicity of CNV syndromes is difficult and often relies on the identification of potential candidates through manual searches of the literature and online resources. We describe here the development of a computational framework to comprehensively search phenotypic information from model organisms and single-gene human hereditary disorders, and thus speed the interpretation of the complex phenotypes of CNV disorders. There are currently more than 5000 human genes about which nothing is known phenotypically but for which detailed phenotypic information for the mouse and/or zebrafish orthologs is available. Here, we present an ontology-based approach to identify similarities between human disease manifestations and the mutational phenotypes in characterized model organism genes; this approach can therefore be used even in cases where there is little or no information about the function of the human genes. We applied this algorithm to detect candidate genes for 27 recurrent CNV disorders and identified 802 gene-phenotype associations, approximately half of which involved genes that were previously reported to be associated with individual phenotypic features and half of which were novel candidates. A total of 431 associations were made solely on the basis of model organism phenotype data. Additionally, we observed a striking, statistically significant tendency for individual disease phenotypes to be associated with multiple genes located within a single CNV region, a phenomenon that we denote as pheno-clustering. Many of the clusters also display statistically significant similarities in protein function or vicinity within the protein-protein interaction network. Our results provide a basis for understanding previously un-interpretable genotype-phenotype correlations in pathogenic CNVs and for mobilizing the large amount of model organism phenotype data to provide insights into human genetic disorders

Molecular mechanism of CHRDL1-mediated X-linked megalocornea in humans and in Xenopus model

Chordin-Like 1 (CHRDL1) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC—with presumably disturbed cornea growth and differentiation—contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injurie

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM

Application of a functional model for the modernization of devices designed to eliminate emergency oil spills

The article describes the stages of creation and composition of the functional model, which can be used for the design of oil spill response devices. The principle of operation of the functional model given in the article and it's graphical scheme are show

Expression analysis and functional characterization of two novel tumorsuppressor candidate genes in mammary carcinoma

Titelblatt Inhaltsverzeichnis, Danksagung 1\. Einleitung 1 1.1 Epidemiologie des Mammakarzinoms 1.2 Tumorgenese 3 1.3 Strategie zur Validierung von Kandidatengenen 7 1.4 Epidemiologie des Mammakarzinoms 8 1.5 Zell-Matrix-Verbindungen 9 1.6 Wnt-Signaltransduktionsweg11 2\. Zielsetzung 14 3\. Material und Methoden 16 3.1 Puffer und Lösungen 16 3.2 Zelllinien 18 3.3 Plasmid-Isolierung 18 3.4 Sequenzierung 19 3.5 LoH-Analyse 21 3.6 Mutationsanalyse 21 3.7 Northern Hybridisierung 25 3.8 RNA in situ Hybridisierung 27 3.9 Poly-A+-ENA-Präparation 32 3.10 cDNA-Synthese 33 3.11 Quantitative PCR (TaqManTM 35 3.12 DANN-Präparation aus Gewebeproben 38 3.13 Proteinchemische Methoden 39 3.14 Zellbiologische Methoden 51 3.15 Statistische Methoden 58 4\. Ergebnisse 4.1 Auswahl und Bestätigung der differentiellen Expression der Kandidatengene auf RNA-Ebene 59 4.2 Prognostische Relevanz von SFRP1 4.3 Funktionelle Analyse von SFRP1 83 5\. Diskussion 90 5.1 Tensin 90 5.2 Secreted frizzled-related protein 1 (SFRP1) 95 6\. Zusammenfassung 108 7\. Summary 110 8\. Literaturverzeichnis 110 9\. Abbildungsverzeichnis 124 10\. Tabellenverzeichnis 125 11\. Lebenslauf 126 12\. Publikationsliste 127 13\. Anhang 129 13.1 Abkürzungsverzeichnis 129 13.2 Histologie und klinisch-pathologische Eigenschaften der verwendeten Gewebeproben 131 13.3 Primer für Mutationsanalyse 132 13.4 Erklärung 135600 Kandidatengene, die möglicherweise an der Entwicklung von gynäkologischen Tumoren beteiligt sind, wurden mit Hilfe eines in silico Ansatzes (eNorthern) identifiziert. Ausgehend von diesen Kandidatengenen wurden 40 Kandidaten für die weitere Validierung ausgewählt und im Rahmen der vorliegenden Dissertation wurden zwei dieser 40 Kandidatengene, Tensin und SFRP1, untersucht. Für beide Kandidatengene konnten die in silico Daten auf RNA-Ebene mit mehreren unabhängigen Methoden (Northern Blot, quantitative PCR, RNA in situ Hybridisierung) bestätigt werden. Beide Gene zeigen in den Tumorzellen von gynäkologischen Karzinomen eine starke Expressionsreduktion im Vergleich zum entsprechenden Normalepithel. Da mit Mutationsanalysen keine Veränderungen der kodierenden Gensequenz nachgewiesen werden konnten, wird postuliert, dass beide Kandidatengene zur Tumorsuppressorgen Klasse II zählen. Als Komponente der Fokalkontakte ist Tensin sowohl an der Zell-Matrix-Adhäsion, als auch an der Signaltransduktion beteiligt. Die Analyse des Tensin-Expressionsmusters in Brustgewebe mit Hilfe der RNA in situ Hybridisierung zeigte eine starke Expression vor allem in Epithelzellen des normalen Brustgewebes. In Brusttumorzellen war die Tensin-Expression hingegen in ca. 50% der untersuchten Fälle reduziert oder vollständig verloren. Diese RNA-Daten stützen die auf den Daten des eNortherns basierende Hypothese, dass Tensin möglicherweise Tumorsuppressorfunktion besitzt. Es konnten keine Sequenzveränderungen in der kodierenden Sequenz des Tensin-Gens in genomischer DNA aus Brusttumorgewebe nachgewiesen werden. Das Tensin-Gen könnte während der Tumorentstehung stattdessen durch epigenetische Mechanismen wie Promotor- Hypermethylierung inaktiviert werden. Ob der Verlust der Tensin-Expression direkte Relevanz für die Tumorentstehung hat oder ob das Kandidatengen in der Tumordiagnostik als Marker verwendet werden kann, muss durch weitere Untersuchungen in Zellkultur-Modellen und auf Proteinebene, z.B. an �Tissue Microarrays�, analysiert werden. Das zweite in dieser Arbeit untersuchte Gen, SFRP1, ist ein negativer Regulator des Wnt-Signaltransduktionsweges, der in der Entstehung von soliden Tumoren, z.B. Brust- und Kolontumoren eine wichtige Rolle spielt. Ein selbst-generierter, polyklonaler SFRP1-Antikörper wurde charakterisiert, um die SFRP1 Expression auf Proteinebene zu analysieren und die Assoziation mit klinischen Parametern und tumorspezifischem Überleben zu untersuchen. Die Analyse von 56 in situ Karzinomen und mehr als 2000 invasiven Karzinomen ergab, dass SFRP1 in diesen Karzinomen im gleichen Maß (43% bzw. 46%) vollständig verloren ist. Das deutet darauf hin, dass der Verlust von SFRP1 ein frühes Ereignis in der Tumorentstehung darstellt. Die SFRP1 Expression ist revers mit der Tumorgröße (pT) korreliert (p < 0,001), aber es wurde keine Korrelation mit anderen klinischen Parametern wie Tumorgrad oder Lymphknotenstatus beobachtet. Dies konnte in einer multivariaten Analyse bezüglich der Assoziation von SFRP1 Expression und Tumorgröße bestätigt werden (p = 0,029). Bei der Korrelation der Überlebensdaten mit der SFRP1 Expression konnte eine schlechtere Prognose für Patientinnen mit SFRP1-negativen kleinen Tumoren (pT1) beobachtet werden (p = 0,04). Die funktionellen Analysen in Brusttumor-Zelllinien ergaben eine mögliche Funktion von SFRP1 in der Regulation der Zelladhäsion. In den hier verwendeten Modellen konnte keine Bedeutung von SFRP1 für die Kontrolle der Invasivität von Tumorzellen beobachtet werden. Ein Einfluss auf die Proliferation von Tumorzelllinien war zu bestimmten Zeitpunkten (24h und 48h) nachweisbar. Folglich ist der Verlust von SFRP1 wahrscheinlich nicht als initialer Faktor für die Tumorentstehung relevant, sondern in Verbindung mit anderen genetischen Veränderungen. SFRP1 könnte aber ein neuer prognostischer Marker in der Brustkrebs- Diagnostik/-Therapie bei frühen Tumoren sein.600 candidate genes which are possibly involved in the development of gynecological tumors were identified using an in silico approach (eNorthern). Fourty of these candidate genes were selected for further validation within a German research consortium. This dissertation project deals with the validation of two candidate genes, Tensin and SFRP1. The in silico data for Tensin and SFRP1 were confirmed on the RNA level by three independent methods (Northern Blot, quantitative PCR and RNA in situ hybridization). Both genes show strongly reduced expression in gynecological tumors compared to normal tissue. Since no aberrations were detected in the coding sequence of both genes, I postulate that the two candidate genes belong to the group of class II tumor suppressor genes. Tensin is part of focal adhesion complexes, thus playing a role in cell-matrix adhesion as well as in signal transduction processes. The analysis of the Tensin expression pattern in mammary gland tissue using RNA in situ hybridization revealed an abundant expression in normal breast epithelial cells. Breast tumor cells exhibited a reduced expression or complete loss of Tensin in ~50% of all cases investigated in this study. The RNA expression data support the possible involvement of Tensin as a tumor suppressor gene in breast cancer development. No mutations were identified in the coding sequence of Tensin in genomic DNA isolated from breast tumor tissues. It is conceivable that the Tensin inactivation during tumor development is due to epigenetic mechanisms, i.e. promoter hypermethylation. If the loss of Tensin expression is relevant for tumorigenesis or if Tensin can be used as a novel marker in cancer diagnostics, has to be shown by further studies on protein level as well as in cell culture experiments. The second gene investigated in this thesis, secreted Frizzled-related protein 1 (SFRP1), is a negative regulator of the Wnt pathway. An SFRP1 specific antibody was generated and characterized to analyze SFRP1 expression on protein level and to investigate the association of SFRP1 expression with clinicopathological parameters and patient survival. The analysis of >2000 invasive breast tumors and 56 carcinoma in situ revealed similar frequencies of SFRP1 loss in these tumors (46% and 43% respectively). Therefore, I propose that loss of SFRP1 expression is an early event in breast tumorigenesis. SFRP1 expression was inversely correlated with tumor stage (p<0.001) but not with other prognostic parameters like tumor grade or lymph node status. Performing a multivariate analysis we could confirm the association between tumor stage and SFRP1 expression (p=0.029). In particular, loss of SFRP1 expression in early stage breast tumors (pT1) was associated with poor prognosis (p=0.04). Functional analyses revealed a possible influence of SFRP1 on the regulation of cell adhesion to the ECM whereas no effect on invasiveness of tumor cell lines was observed in the cell culture model. A possible involvement in proliferation of tumor cell was detectable at certain time points (24h and 48h). In conclusion, loss of SFRP1 is most likely not the initial event directly leading to breast tumorigenesis but facilitating breast cancer development if other genetic changes occur in the cell. Still, SFRP1 expression might be useful as a novel prognostic marker in early stage breast cancer

Left ventricular hypertrabeculation/noncompaction with epilepsy, other heart defects, minor facial anomalies and new copy number variants

Abstract Background Left ventricular hypertrabeculation/noncompaction (LVHT) is a cardiac abnormality of unknown etiology which has been described in children as well as in adults with and without chromosomal aberrations. LVHT has been reported in association with various cardiac and extracardiac abnormalities like epilepsy and facial dysmorphism. Case presentation A unique combination of LVHT, atrial septal defect, pulmonary valve stenosis, aortic stenosis, epilepsy and minor facial anomalies is presented in a 5.5 years old girl. Microarray-based genomic hybridization (array-CGH) detected six previously not described copy number variants (CNVs) inherited from a clinically unaffected father and minimally affected mother, thus, most likely, not clinically significant but rare benign variants. Conclusions Despite this complex phenotype de novo microdeletions or microduplications were not detected by array CGH. Further investigations, such as whole exome sequencing, could reveal point mutations and small indels as the possible cause.</p