Boosting care and knowledge about hereditary cancer: European Reference Network on Genetic Tumour Risk Syndromes.

Approximately 27-36 million patients in Europe have one of the ~ 5.000-8.000 known rare diseases. These patients often do not receive the care they need or they have a substantial delay from diagnosis to treatment. In March 2017, twenty-four European Reference Networks (ERNs) were launched with the aim to improve the care for these patients through cross border healthcare, in a way that the medical knowledge and expertise travels across the borders, rather than the patients. It is expected that through the ERNs, European patients with a rare disease get access to expert care more often and more quickly, and that research and guideline development will be accelerated resulting in improved diagnostics and therapies. The ERN on Genetic Tumour Risk Syndromes (ERN GENTURIS) aims to improve the identification, genetic diagnostics, prevention of cancer, and treatment of European patients with a genetic predisposition for cancer. The ERN GENTURIS focuses on syndromes such as hereditary breast cancer, hereditary colorectal cancer and polyposis, neurofibromatosis and more rare syndromes e.g. PTEN Hamartoma Tumour Syndrome, Li Fraumeni Syndrome and hereditary diffuse gastric cancer

One at a time, live tracking of NGF axonal transport using quantum dots

Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50–150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body

Reversible thrombocytopenia during hibernation originates from storage and release of platelets in liver sinusoids

Immobility is a risk factor for thrombosis due to low blood flow, which may result in activation of the coagulation system, recruitment of platelets and clot formation. Nevertheless, hibernating animals-who endure lengthy periods of immobility-do not show signs of thrombosis throughout or after hibernation. One of the adaptations of hemostasis in hibernators consists of a rapidly reversible reduction of the number of circulating platelets during torpor, i.e., the hibernation phase with reduction of metabolic rate, low blood flow and immobility. It is unknown whether these platelet dynamics in hibernating hamsters originate from storage and release, as suggested for ground squirrel, or from breakdown and de novo synthesis. A reduction in detaching forces due to low blood flow can induce reversible adhesion of platelets to the vessel wall, which is called margination. Here, we hypothesized that storage-and-release by margination to the vessel wall induces reversible thrombocytopenia in torpor. Therefore, we transfused labeled platelets in hibernating Syrian hamster (Mesocricetus auratus) and platelets were analyzed using flow cytometry and electron microscopy. The half-life of labeled platelets was extended from 20 to 30 h in hibernating animals compared to non-hibernating control hamsters. More than 90% of labeled platelets were cleared from the circulation during torpor, followed by emergence during arousal which supports storage-and-release to govern thrombocytopenia in torpor. Furthermore, the low number of immature platelets, plasma level of interleukin-1α and normal numbers of megakaryocytes in bone marrow make platelet synthesis or megakaryocyte rupture via interleukin-1α unlikely to account for the recovery of platelet counts upon arousal. Finally, using large-scale electron microscopy we revealed platelets to accumulate in liver sinusoids, but not in spleen or lung, during torpor. These results thus demonstrate that platelet dynamics in hibernation are caused by storage and release of platelets, most likely by margination to the vessel wall in liver sinusoids. Translating the molecular mechanisms that govern platelet retention in the liver, may be of major relevance for hemostatic management in (accidental) hypothermia and for the development of novel anti-thrombotic strategies

Flexible and Extended Linker Domains Support Efficient Targeting of Heh2 to the Inner Nuclear Membrane

The nuclear pore complex (NPC) is embedded in the nuclear envelope and forms the main gateway to the nuclear interior including the inner nuclear membrane (INM). Two INM proteins in yeast are selectively imported. Their sorting signals consist of a nuclear localization signal, separated from the transmembrane domain by a long intrinsically disordered (ID) linker. We used computational models to predict the dynamic conformations of ID linkers and analyzed the INM targeting efficiency of proteins with linker regions with altered Stokes radii and decreased flexibilities. We find that flexibility, Stokes radius, and the frequency at which the linkers are at an extended end-to-end distance larger than 25 nm are good predictors for the targeting of the proteins. The data are consistent with a transport mechanism in which INM targeting of Heh2 is dependent on an ID linker that facilitates the crossing of the approximately 25-nm thick NPC scaffold

Improvements in Spelling after QEEG-based Neurofeedback in Dyslexia: A Randomized Controlled Treatment Study

Phonological theories of dyslexia assume a specific deficit in representation, storage and recall of phonemes. Various brain imaging techniques, including qEEG, point to the importance of a range of areas, predominantly the left hemispheric temporal areas. This study attempted to reduce reading and spelling deficits in children who are dyslexic by means of neurofeedback training based on neurophysiological differences between the participants and gender and age matched controls. Nineteen children were randomized into an experimental group receiving qEEG based neurofeedback (n = 10) and a control group (n = 9). Both groups also received remedial teaching. The experimental group improved considerably in spelling (Cohen’s d = 3). No improvement was found in reading. An indepth study of the changes in the qEEG power and coherence protocols evidenced no fronto-central changes, which is in line with the absence of reading improvements. A significant increase of alpha coherence was found, which may be an indication that attentional processes account for the improvement in spelling. Consideration of subtypes of dyslexia may refine the results of future studies

LED Arrays as Cost Effective and Efficient Light Sources for Widefield Microscopy

New developments in fluorophores as well as in detection methods have fueled the rapid growth of optical imaging in the life sciences. Commercial widefield microscopes generally use arc lamps, excitation/emission filters and shutters for fluorescence imaging. These components can be expensive, difficult to maintain and preclude stable illumination. Here, we describe methods to construct inexpensive and easy-to-use light sources for optical microscopy using light-emitting diodes (LEDs). We also provide examples of its applicability to biological fluorescence imaging

In-Cell Biochemistry Using NMR Spectroscopy

Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry

HIF-1 and c-Src Mediate Increased Glucose Uptake Induced by Endothelin-1 and Connexin43 in Astrocytes

In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism

Development of FRET Assay into Quantitative and High-throughput Screening Technology Platforms for Protein–Protein Interactions

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein–protein interactions. Here we report the development of dissociation constant (Kd) determination for protein–protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction Kd determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The Kd determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both Kd determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications