Genome transcription/translation of segmented, negative-strand RNA viruses

The requirements for alignment of capped RNA leader sequences along the viral genome during influenza transcription initiation (“cap-snatching”) have long been an enigma. Previous work on Tomato spotted wilt virus (TSWV) transcription initiation has revealed that this virus displays a preference for leaders with increasing base complementarity to the 3'-ultimate residues of the viral RNA template. Assuming that cap-snatching is a highly conserved mechanism, it is tempting to speculate that the findings for TSWV apply to all segmented negative RNA viruses. The research in this thesis aimed to analyze whether similar cap donor requirements applied for Influenza A virus transcription initiation as compared to what has been found for TSWV. Indeed, in vitro studies demonstrated that influenza transcriptase prefers multiple base-pairing capped leaders. Additionally, the occurrence of “prime-and-realign” during influenza transcription initiation was observed, as well as internal priming at the 3'-penultimate viral residue. The in vitro findings were confirmed by similar studies performed during influenza infection of cell cultures. Whereas transcription initiation of TSWV has been relatively well studied, transcription termination has not. It is postulated that transcription termination/translation is triggered by the formation of a hairpin structure. In cell experiments support a role of the TSWV hairpin structure in translation.</p

Principles guiding embryo selection following genome-wide haplotyping of preimplantation embryos.

STUDY QUESTION How to select and prioritize embryos during PGD following genome-wide haplotyping? SUMMARY ANSWER In addition to genetic disease-specific information, the embryo selected for transfer is based on ranking criteria including the existence of mitotic and/or meiotic aneuploidies, but not carriership of mutations causing recessive disorders. WHAT IS KNOWN ALREADY Embryo selection for monogenic diseases has been mainly performed using targeted disease-specific assays. Recently, these targeted approaches are being complemented by generic genome-wide genetic analysis methods such as karyomapping or haplarithmisis, which are based on genomic haplotype reconstruction of cell(s) biopsied from embryos. This provides not only information about the inheritance of Mendelian disease alleles but also about numerical and structural chromosome anomalies and haplotypes genome-wide. Reflections on how to use this information in the diagnostic laboratory are lacking. STUDY DESIGN, SIZE, DURATION We present the results of the first 101 PGD cycles (373 embryos) using haplarithmisis, performed in the Centre for Human Genetics, UZ Leuven. The questions raised were addressed by a multidisciplinary team of clinical geneticist, fertility specialists and ethicists. PARTICIPANTS/MATERIALS, SETTING, METHODS Sixty-three couples enrolled in the genome-wide haplotyping-based PGD program. Families presented with either inherited genetic variants causing known disorders and/or chromosomal rearrangements that could lead to unbalanced translocations in the offspring. MAIN RESULTS AND THE ROLE OF CHANCE Embryos were selected based on the absence or presence of the disease allele, a trisomy or other chromosomal abnormality leading to known developmental disorders. In addition, morphologically normal Day 5 embryos were prioritized for transfer based on the presence of other chromosomal imbalances and/or carrier information. LIMITATIONS, REASONS FOR CAUTION Some of the choices made and principles put forward are specific for cleavage-stage-based genetic testing. The proposed guidelines are subject to continuous update based on the accumulating knowledge from the implementation of genome-wide methods for PGD in many different centers world-wide as well as the results of ongoing scientific research. WIDER IMPLICATIONS OF THE FINDINGS Our embryo selection principles have a profound impact on the organization of PGD operations and on the information that is transferred among the genetic unit, the fertility clinic and the patients. These principles are also important for the organization of pre- and post-counseling and influence the interpretation and reporting of preimplantation genotyping results. As novel genome-wide approaches for embryo selection are revolutionizing the field of reproductive genetics, national and international discussions to set general guidelines are warranted. STUDY FUNDING/COMPETING INTEREST(S) The European Union's Research and Innovation funding programs FP7-PEOPLE-2012-IAPP SARM: 324509 and Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D. and M.Z.E. J.R.V., T.V. and M.Z.E. have patents ZL910050-PCT/EP2011/060211-WO/2011/157846 (‘Methods for haplotyping single cells’) with royalties paid and ZL913096-PCT/EP2014/068315-WO/2015/028576 (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’) with royalties paid, licensed to Cartagenia (Agilent technologies). J.R.V. also has a patent ZL91 2076-PCT/EP20 one 3/070858 (‘High throughout genotyping by sequencing’) with royalties paid

Cardiovascular risk in chronic myeloid leukaemia: A multidisciplinary consensus on screening and management

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) have become the mainstay of treatment for chronic myeloid leukaemia (CML), but cardiovascular (CV) risk and exacerbation of underlying risk factors associated with TKIs have become widely debated. Real-world evidence reveals little application of CV risk factor screening or continued monitoring within UK CML management. This consensus paper presents practical recommendations to assist healthcare professionals in conducting CV screening/comorbidity management for patients receiving TKIs. METHODS: We conducted a multidisciplinary panel meeting and two iterative surveys involving 10 CML specialists: five haematologists, two cardio-oncologists, one vascular surgeon, one haemato-oncology pharmacist and one specialist nurse practitioner. RESULTS: The panel recommended that patients commencing second-/third-generation TKIs undergo formal CV risk assessment at baseline, with additional investigations and involvement of cardiologists/vascular surgeons for those with high CV risk. During treatment, patients should undergo CV monitoring, with the nature and frequency of testing dependent on TKI and baseline CV risk. For patients who develop CV adverse events, decision-making around TKI interruption, cessation or change should be multidisciplinary and balance CV and haematological risk. CONCLUSION: The panel anticipates these recommendations will support healthcare professionals in implementing CV risk screening and monitoring, broadly and consistently, and thereby help optimise TKI treatment for CML

Towards elucidating carnosic acid biosynthesis in Lamiaceae: Functional characterization of the three first steps of the pathway in Salvia fruticosa and Rosmarinus officinalis

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPSand SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera

Combining macula clinical signs and patient characteristics for age-related macular degeneration diagnosis: a machine learning approach

Background: To investigate machine learning methods, ranging from simpler interpretable techniques to complex (non-linear) “black-box” approaches, for automated diagnosis of Age-related Macular Degeneration (AMD). Methods: Data from healthy subjects and patients diagnosed with AMD or other retinal diseases were collected during routine visits via an Electronic Health Record (EHR) system. Patients’ attributes included demographics and, for each eye, presence/absence of major AMD-related clinical signs (soft drusen, retinal pigment epitelium, defects/ pigment mottling, depigmentation area, subretinal haemorrhage, subretinal fluid, macula thickness, macular scar, subretinal fibrosis). Interpretable techniques known as white box methods including logistic regression and decision trees as well as less interpreitable techniques known as black box methods, such as support vector machines (SVM), random forests and AdaBoost, were used to develop models (trained and validated on unseen data) to diagnose AMD. The gold standard was confirmed diagnosis of AMD by physicians. Sensitivity, specificity and area under the receiver operating characteristic (AUC) were used to assess performance. Results: Study population included 487 patients (912 eyes). In terms of AUC, random forests, logistic regression and adaboost showed a mean performance of (0.92), followed by SVM and decision trees (0.90). All machine learning models identified soft drusen and age as the most discriminating variables in clinicians’ decision pathways to diagnose AMD. C Conclusions: Both black-box and white box methods performed well in identifying diagnoses of AMD and their decision pathways. Machine learning models developed through the proposed approach, relying on clinical signs identified by retinal specialists, could be embedded into EHR to provide physicians with real time (interpretable) support

Diagnosis of Pancreatic Ductal Adenocarcinoma and Chronic Pancreatitis by Measurement of microRNA Abundance in Blood and Tissue

A solid process for diagnosis could have a substantial impact on the successful treatment of pancreatic cancer, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analyzed on microarrays and in part validated by real-time PCR assays. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors. Utilizing the minimally invasive blood test, receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analysis demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis; respective AUC values of 0.973 and 0.950 were obtained. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples with AUC values of 1.0 and 0.937, respectively. In addition, discrimination between cancer and chronic pancreatitis was achieved (AUC = 0.875). Also, several miRNAs were identified that exhibited abundance variations in both tissue and blood samples. The results could have an immediate diagnostic value for the evaluation of tumor reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer