Sequential improvement in paediatric medulloblastoma outcomes in a low-and-middle-income country setting over three decades

Background: Medulloblastoma (MB) is the commonest malignant brain tumour of childhood. Accurate clinical data for paediatric MB in the LMIC setting is lacking. Sequential improvements in outcome seen in high income countries are yet to be reflected in LMIC. Aim: Quantification of paediatric MB outcomes in the LMIC setting over three decades of advances in multidisciplinary intervention. Setting: Cape Town, South Africa Methods: This was a retrospective study of 136 children with MB diagnosed between 1985 and 2015. Modified Chang criteria were used for risk stratification. The primary study objective was overall survival (OS), quantified by analysis of epidemiological, clinical and pathological data. Results: OS improved significantly during the most recent decade (2005-2015) when compared with the preceding two decades (1985-1995 and 1995-2005). Despite reduced dose craniospinal irradiation for standard risk cases, OS was significantly greater than during the preceding two decades. High-risk disease was identified in 71.4% of cases and was associated with significantly inferior OS compared with standard risk cases. Improved OS was positively correlated with therapeutic era, 3-D conformal radiotherapy technique, older age at diagnosis, classic and desmoplastic histology, extent of resection and absence of leptomeningeal spread on imaging. Conclusion: Advances in multidisciplinary management of MB in our combined service are associated with improved survival. Access to improved imaging modalities, advances in surgical techniques, increased number of patients receiving risk-adapted combination chemo- and/or radiotherapy as well as craniospinal irradiation using a linear accelerator with 3D planning, are considered as contributing factors

Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

<p>Abstract</p> <p>Background</p> <p>Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.</p> <p>Results</p> <p>During a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP).</p> <p>To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor.</p> <p>We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by <it>in vivo </it>co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage.</p> <p>Conclusions</p> <p>This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important regulator of cardiac contractility. This has further implications for understanding the patho-aetiology of HCM-causing mutations in the genes encoding cMyBPC and cTNI, and raises the question of whether MMGL might itself be considered a candidate HCM-causing or modifying factor.</p

The type 1 insulin-like growth factor receptor (IGF1R) as anti-cancer treatment target.

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Functional analysis and recombinant expression of a sea urchin G-string binding factor

Part of work presented in this thesis has been published: Regulation of gene expressions by GC-rich DNA cis-elements / J.P. Hapgood, J. Riedemann and S.D. Scherer in Cell biology international, vol. 25, 2001.Thesis (MSc)--Stellenbosch University, 2001.ENGLISH ABSTRACT: The sea urchin G-string binding factor 1 (suGF1) has previously been shown to bind with high affinity and selectivity to stretches of contiguous deoxyguanosine residues, a DNA motif found in the upstream regions of many unrelated genes from several organisms. It has been proposed that suGF1 plays a role in transcriptional regulation. Homopurine.homopyrimidine stretches have been shown to form unusual DNA structures, in vitro. To investigate the potential of the suGF1 binding site to form unusual structures under certain conditions, synthetic oligodeoxyribonucleotides containing the suGF1 poly(dG).(dC) binding site were subjected to circular dichroism (CD) analyses. The CD results indicate that the suGF1 binding site forms a mixture of unusual DNA structures, as deduced by comparison with the spectra obtained for B-DNA, triplex and quadruplex conformations. These results are consistent with the hypothesis that suGF1 specifically recognises G-strings that exhibit unusual structures. Exhaustive database searches showed that suGF1 has no significant homology with any previously identified proteins or cDNAs from any species. Given the relevance of mammalian models to medical science, and since no sea urchin cell lines are currently available, the identification of a mammalian functional homologue would facilitate determination of the in vivo function of such a potentially important, putative, novel DNAbinding protein in mammalian cell lines. In this study sequence analysis tools were used to identify hORFX, a putative human functional homologue of suGF1. Similarities in the domain organisation of the two proteins, prompted an investigation into the DNA-binding properties of hORFX, as well as a more detailed structure prediction analysis, with a view to determining whether hORFX is a functional homologue of suGF1. hORFX was successfully expressed in vitro, but lacked the ability to specifically bind G-strings. Theoretical predictions suggest that suGF1 has a DNA-binding domain belonging to a different family to that predicted for hORFX, consistent with differences in their respective DNA-binding specificities. suGF1 and hORFX were predicted to have helix-turn-helix and helix-loop-helix DNA-binding domains, respectively. Taken together the results do not support the hypothesis that hORFX is a suGF1 homologue. To date, no direct evidence for the in vivo function of suGF1 has been obtained. With a view to performing transactivation assays in the future, the expression of suGF1 in yeast was investigated in this project. An suGF1 expression construct was engineered and transformed into a protease-deficient yeast strain. Nuclear extracts were prepared and subjected to SOS-PAGE and electrophoretic mobility shift assays (EMSAs). suGF1 was shown to be successfully expressed in yeast cells and exhibited similar G-string-binding properties to that of native and in vitro transcribed and translated (IVT) suGF1. The suGF1 eDNA was also subjected to in si/ico expression, which together with the SDSPAGE results of yeast nuclear extracts and IVT suGF1, indicated that the protein might be expressed as multiple truncated products, due to the utilisation of multiple AUG translation start sites. These in vitro results are crucial for the ultimate outcome and correct interpretation of future transactivation experiments and lay the foundation for further investigation into the possible role of suGF1 in transcriptional regulation.AFRIKAANSE OPSOMMING: In die verlede is bewys dat die seepampoentjie G-string-bindende faktor (suGF1) hoë affiniteit en spesifisiteit vir aaneenlopende volgordes van deoksiguanosien residue besit. Hierdie DNA motief kom algemeen voor in die stroom-op gebiede van verskeie gene in verskillende organismes. Daar is 'n veronderstelling dat suGF1 betrokke is by die regulering van geenuitdrukking. Vroeër is bewys dat homopurien.homopirimidien-ryke areas die vermoë besit om in vitro ongewone DNA-strukture te vorm. Die potentiaal van die suGF1-bindingsetel om ongewone DNA-strukture te vorm is gevolglik deur sirkulêre dikroïsme (SD) analise ondersoek. Vergelyking van die spektra vir B-DNA-, tripleks- en kwadrupleks-strukture met dié van die suGF1-bindingsetel, toon duidelik dat laasgenoemde 'n mengsel van ongewone DNA konformasies, onder die spesifieke eksperimentele omstandigehede, aanneem. Deeglike inspeksie van die beskikbare geen- en proteïendatabasisse vir alle spesies het aangetoon dat suGF1 geen merkbare kDNA- of proteïenhomoloë besit nie. As gevolg van die belang van soogdiermodelsisteme in die mediese wetenskappe, asook die onbeskikbaarheid van seepampoentjie-sellyne, is 'n soektog na 'n funktionele suGF1 homoloog in soogdiere geloods. Die ontdekking van só 'n homoloog sal dit moontlik maak om die rol van hierdie potensiaal belangrike en unieke DNA-bindingsproteïen te ondersoek. Tydens hierdie soektog is spesiale analise-programme gebruik en 'n potensiële menshomoloog van suGF1, hORFX, is geïdentifiseer. Die mees prominente ooreenkoms tussen die twee proteïene is die soortgelyke rangskikking van funksionele motiewe. Gevolglik is die DNA-bindings eienskappe van die hORFX-proteïen ondersoek, insluitende 'n detaileerde struktuur-funksie-voorspelling ten einde vas te stel of dit wél 'n homoloog van suGF1 is. hORFX is suksesvol uitgedruk in vitro, maar besit nie die vermoë om dieselfde G-string waaraan suGF1 spesifiek bind te herken nie. Teoretiese analise het voorspel dat suGF1 en hORFX aan verskillende DNA-bindings proteïen-families behoort, aangesien suGF1 'n heliks-draai-heliks en hORFX 'n heliks-lus-heliks motief bevat. Hierdie inligting, tesame met die eksperimentele resultate, dui aan dat hORFX nie 'n homoloog van suGF1 is nie. Tot op hede is daar niks bekend aangaande suGF1 se funksie in vivo nie. Met die oog op transaktiveringseksperimente in die toekoms, is die ekspressie van suGF1 in gisselle tydens hierdie navorsingsprojek ondersoek. 'n suGF1 ekspressievektor is berei en gebruik om 'n protease-negatiewe gissellyn te transformeer. Kernekstrakte is ondersoek deur SDS-PAGE en elektroforetiese mobiliteitsessais. Daar is gevind dat suGF1 suksesvol uitgedruk is in die gisselle. Die rekombinante suGF1 besit G-volgorde bindingsaktiwiteite soortgelyk aan dié van suGF1 in kernekstrakte van seepampoentjies, asook in vitro getranskribeerde-en getransleerde suGF1. Die kDNA vir suGF1 is ook in silico uitgedruk. Tesame met die SDS-PAGE-resultate het laasgenoemde aangetoon dat die suGF1-kDNA veelvuldige AUG-kodons bevat vir die inisiasie van proteïentranslasie. Dit lei moontlik tot die translasie van 'n reeks proteïenprodukte wat verkort is aan die N-terminale kant, afgesien van die volledige suGF1-proteïen. Die in vitro resultate in geheel is essensieel vir die toekomstige uitvoering en interpretasie van transaktiveringseksperimente. Hierdie projek lê gevolglik die fondasie vir 'n verdere ondersoek na die rol van suGF1 in die regulering van geenuitdrukking

A simple and cost-effective method for producing small interfering RNAs with high efficacy

Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity. This approach is expensive due to the cost of chemical synthesis of RNAs. We report a technically simple, quick and cost-effective method for the production of siRNAs that makes use of in vitro transcription and deoxyribozyme digestion of the transcripts to produce the desired sequence and length. The method allows for several siRNAs to be produced in parallel at much reduced costs. The siRNAs produced with this method were tested in MDA-MB-231 human breast cancer cells for efficacy against the type 1 insulin-like growth factor receptor (IGF1R) mRNA and they caused dose-dependent inhibition of IGF1R expression comparable to that induced by chemically synthesised siRNAs of the same sequence. This method is also useful for producing long RNA fragments of defined length and sequence that may be difficult to synthesise chemically, and also for producing large quantities of RNAs for applications including structural studies and the study of interactions between RNA and other molecules, such as proteins, other nucleic acids and drugs

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells

Abstract Background The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high‐grade prostate cancers. We describe a non‐invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD‐CTCs) from blood samples followed by their identification by immunostaining for pan‐cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl‐acyl coenzyme‐A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). Methods Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case–control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. Results Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. Conclusions The test accurately detects PrAD‐CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection