Kinome and mRNA expression profiling of high-grade osteosarcoma cell lines implies Akt signaling as possible target for therapy

Background: High-grade osteosarcoma is a primary malignant bone tumor mostly occurring in adolescents and young adults, with a second peak at middle age. Overall survival is approximately 60%, and has not significantly increased since the introduction of neoadjuvant chemotherapy in the 1970s. The genomic profile of high-grade osteosarcoma is complex and heterogeneous. Integration of different types of genome-wide data may be advantageous in extracting relevant information from the large number of aberrations detected in this tumor. Methods: We analyzed genome-wide gene expression data of osteosarcoma cell lines and integrated these data with a kinome screen. Data were analyzed in statistical language R, using LIMMA for detection of differential expression/phosphorylation. We subsequently used Ingenuity Pathways Analysis to determine deregulated pathways in both data types. Results: Gene set enrichment indicated that pathways important in genomic stability are highly deregulated in these tumors, with many genes showing upregulation, which could be used as a prognostic marker, and with kinases phosphorylating peptides in these pathways. Akt and AMPK signaling were identified as active and inactive, respectively. As these pathways have an opposite role on mTORC1 signaling, we set out to inhibit Akt kinases with the allosteric Akt inhibitor MK-2206. This resulted in inhibition of proliferation of osteosarcoma cell lines U-2 OS and HOS, but not of 143B, which harbors a KRAS oncogenic transformation. Conclusions: We identified both overexpression and hyperphosphorylation in pathways playing a role in genomic stability. Kinome profiling identified active Akt signaling, which could inhibit proliferation in 2/3 osteosarcoma cell lines. Inhibition of PI3K/Akt/mTORC1 signaling may be effective in osteosarcoma, but further studies are required to determine whether this pathway is active in a substantial subgroup of this heterogeneous tumor

Rapid screening for chromosomal aneuploidies using array-MLPA

<p>Abstract</p> <p>Background</p> <p>Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening.</p> <p>Methods</p> <p>We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes.</p> <p>Results</p> <p>In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases.</p> <p>Conclusions</p> <p>Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.</p

The experience of adolescents and parents before, after, and in the process of redirection into the Adapted program of education with lower educational standard

This diploma paper studies the experience of adolescents and their parents where the adolescents were redirected from the regular school program into the Adapted program of education with lower educational standard. Individuals with mild intellectual disability are typically characterised by reduced learning abilities, but in the adapted learning environment they are able to achieve basic school aims. Due to extensive learning difficulties such students are unable to follow the regular school program, which is why they are schooled, or are later redirected into the Adapted program of education with lower educational standard. If the students are redirected later, learning difficulties and failure in the field of education increase, which can cause behavioural and emotional problems, as well as have an important role on students' self-respect. Due to constant failure the students may develop fear of school, and consequently dislike of learning or of school in general. Emotional tension and distress related to school can cause psychosomatic problems or disorders in students. But learning failure extends beyond the scope of student's perception of school alone. It also affects how students spend their free time and their social relations in the broader and narrow environment. This research studies four students, who were redirected into the Adapted program of education with lower educational standard. It is a case study which provides an insight into the experience of parents and their children in the process of redirection into the above mentioned program. To obtain the research data we used the half-structured interview. The interviews were recorded and transcribed. The transcript was then summarized and paraphrased and then analysed in accordance with elements of qualitative text analysis. The research shows that the students felt unsuccessful in the regular school, having difficulties in their relations with peers and teachers, as well as spending a lot of time for their school work. Their parents’ observations were similar, reporting about various behavioural and emotional problems of their children. After the redirection both the students and their parents noted that they had less learning difficulties, the students' well-being improved along with their attitude to school and relations with teachers and students. This research can help understand the problems of students with learning difficulties that are, despite effort and assistance, unable to achieve minimal learning standards. The experience of students and parents is a source of information to those parents and teachers who are dealing with the process of redirection into the Adapted program of education with lower educational standard

Pulmonary Vein, Dorsal Atrial Wall and Atrial Septum Abnormalities in Podoplanin Knockout Mice With Disturbed Posterior Heart Field Contribution

The developing sinus venosus myocardium, derived from the posterior heart field, contributes to the atrial septum, the posterior atrial wall, the sino-atrial node, and myocardium lining the pulmonary and cardinal veins, all expressing podoplanin, a coelomic and myocardial marker. . We compared development and differentiation of the myocardium and vascular Wall of the pulmonary veins (PV), left atrial dorsal wall, and atrial septum in wild type with podoplanin knockout mouse embryos (E10.5-E18.5) by 3D reconstruction and immunohistochemistry. Expression of Nkx2.5 in the pulmonary venous myocardium changes from mosaic to positive during development pointing Out a high proliferative rate compared with Nkx2.5 negative myocardium of the sino-atrial node and cardinal veins. In Mutants, myocardium of the PVs, dorsal atrial wall and atrial septum was hypoplastic. The atrial septum and right-sided wall of the PV almost lacked interposed mesenchyme. Extension of smooth muscle cells into the left atrial body was diminished. We conclude that myocardium of the PVs, dorsal atrial wall, and atrial septum, as well as the smooth Muscle cells, are derived from the posterior heart field regulated by podoplanin. (Pediatr Res 65: 27-32, 2009