MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities

CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM

Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity

Biological Evaluation of Uridine Derivatives of 2-Deoxy Sugars as Potential Antiviral Compounds against Influenza A Virus

Influenza virus infection is a major cause of morbidity and mortality worldwide. Due to the limited ability of currently available treatments, there is an urgent need for new anti-influenza drugs with broad spectrum protection. We have previously shown that two 2-deoxy sugar derivatives of uridine (designated IW3 and IW7) targeting the glycan processing steps during maturation of viral glycoproteins show good anti-influenza virus activity and may be a promising alternative approach for the development of new anti-influenza therapy. In this study, a number of IW3 and IW7 analogues with different structural modifications in 2-deoxy sugar or uridine parts were synthesized and evaluated for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay in vitro, we showed that compounds 2, 3, and 4 exerted the most inhibitory effect on influenza virus A/ostrich/Denmark/725/96 (H5N2) infection in Madin-Darby canine kidney (MDCK) cells, with 50% inhibitory concentrations (IC50) for virus growth ranging from 82 to 100 (μM) without significant toxicity for the cells. The most active compound (2) showed activity of 82 μM with a selectivity index value of 5.27 against type A (H5N2) virus. Additionally, compound 2 reduced the formation of HA glycoprotein in a dose-dependent manner. Moreover, an analysis of physicochemical properties of studied compounds demonstrated a significant linear correlation between lipophilicity and antiviral activity. Therefore, inhibition of influenza A virus infection by conjugates of uridine and 2-deoxy sugars is a new promising approach for the development of new derivatives with anti-influenza activities

Anti-Hepatitis C Virus Activity of Uridine Derivatives of 2-Deoxy Sugars

Hepatitis C virus (HCV), the etiological agent of the most common and dangerous diseases of the liver, is a major health problem worldwide. Despite many attempts, there is still no vaccine available. Although many drugs have been approved for use mostly in combination regimen, their high costs make them out of reach in less developed regions. Previously, we have synthesized a series of compounds belonging to uridine derivatives of 2-deoxy sugars and have proved that some of them possess antiviral activity against influenza A virus associated with N-glycosylation inhibition. Here, we analyze the antiviral properties of these compounds against HCV. Using cell culture-derived HCV (HCVcc), HCV pseudoparticles (HCVpp), and replicon cell lines, we have shown high anti-HCV activity of two compounds. Our results indicated that compounds 2 and 4 significantly reduced HCVcc propagation with IC50 values in low μM range. Further experiments using the HCVpp system confirmed that both compounds significantly impaired the infectivity of produced HCVpp due to the inhibition of the correct maturation of viral glycoproteins. Overall, our results suggest that inhibiting the glycosylation process might be a good target for new therapeutics not only against HCV, but other important viral pathogens which contain envelopes with highly glycosylated proteins

A network comprising short and long noncoding RNAs and RNA helicase controls mouse retina architecture

Brain regions, such as the cortex and retina, are composed of layers of uniform thickness. The molecular mechanism that controls this uniformity is not well understood. Here we show that during mouse postnatal development the timed expression of Rncr4, a retina-specific long noncoding RNA, regulates the similarly timed processing of pri-miR-183/96/182, which is repressed at an earlier developmental stage by RNA helicase Ddx3x. Shifting the timing of mature miR-183/96/182 accumulation or interfering with Ddx3x expression leads to the disorganization of retinal architecture, with the photoreceptor layer being most affected. We identify Crb1, a component of the adhesion belt between glial and photoreceptor cells, as a link between Rncr4-regulated miRNA metabolism and uniform retina layering. Our results suggest that the precise timing of glia–neuron interaction controlled by noncoding RNAs and Ddx3x is important for the even distribution of cells across layers.ISSN:2041-172

Antiviral Activity of Uridine Derivatives of 2-Deoxy Sugars against Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV

Praktijkonderzoek als professionele leerstrategie in onderwijs en opleiding

In deze publicatie beschrijven we onze visie op praktijkonderzoek als professionele leerstrategie, waarmee beginnende en ervaren docenten hun eigen dagelijkse praktijk kunnen verbeteren. Aan de hand van voorbeelden laten we zien wat praktijkonderzoek kan betekenen in scholen en (leraren)opleidingen en op welke wijze dit vorm kan krijgen. Inhoudsopgave publicatie 'Praktijkonderzoek als professionele leerstrategie in onderwijs en opleiding' 1. Inleiding over doel en introductie van de verschillende hoofdstukken (leeswijzer). 2. Praktijkonderzoek als professionele leerstrategie (Sanneke Bolhuis) Het doel van praktijkonderzoek is met meer inzicht en begrip handelen in de specifieke eigen context. Praktijkonderzoek is daarbij zelf een interventie. Docenten als onderzoekers van eigen praktijk zijn onderdeel van wat zij onderzoeken. 3. Toetsing en beoordelen in het onderwijs: een continu onderzoek naar de ontwikkeling van de leerlingen of studenten (Desirée Joosten-ten Brinke) Leraren en opleiders toetsen de leervorderingen van hun leerlingen en studenten voortdurend. Daarmee zijn ze in feite voortdurend onderzoek aan het doen, ook al zijn ze zich daarvan misschien niet zo bewust. 4. Beter onderbouwd handelen door praktijkonderzoek: voorbeelden en voorwaarden (Ilona Mathijsen) Dit hoofdstuk laat zien hoe een docent door praktijkonderzoek zijn handelen beter of anders onderbouwt en daardoor mogelijk ook beter of anders gaat handelen. 5. Praktijkonderzoek doe je samen (Quinta Kools) Samen praktijkonderzoek doen zorgt voor een betere kwaliteit van het praktijkonderzoek, voor continuïteit, verbondenheid tussen docenten en een groter draagvlak voor de uitkomsten. 6. Het betrekken van belanghebbenden bij praktijkonderzoek (Quinta Kools) In dit hoofdstuk gaat het over de mogelijke betrokkenen of belanghebbenden bij praktijkonderzoek: wie maken deel uit van het probleem, wie zouden een bijdrage kunnen leveren aan het beter begrijpen of een betere aanpak, voor wie heeft het onderzoek mogelijk consequenties? 7. Praktijkonderzoek doen moet je leren (Sanneke Bolhuis) Doel van dit hoofdstuk is om een beeld te geven van de persoonlijke kwaliteiten en competenties die nodig zijn voor het doen van praktijkonderzoek. Ook wordt besproken waar scholen op zouden moeten letten als ze scholing voor praktijkonderzoek door leraren de school in halen. 8. Praktijkonderzoek van studenten in de lerarenopleiding begeleiden (Karen Krol) Dit hoofdstuk bespreekt hoe studenten leren om praktijkonderzoek uit te voeren als professionele leerstrategie en wat dat vraagt van de begeleiders (opleiders in het instituut en/of de school). 9. De rol van leidinggevenden bij praktijkonderzoek in onderwijs en opleiding (Sanneke Bolhuis en Quinta Kools). In dit hoofdstuk worden de condities besproken die leidinggevenden moeten creëren om praktijkonderzoek tot de 'normale' onderwijspraktijk te laten behoren. Intermezzo's Tussen de hoofdstukken worden korte voorbeelden van praktijkonderzoek beschreven. Dit zijn voorbeelden van praktijkonderzoek door: o lerarenopleiders, die onderzoek deden in een kenniskring van het lectoraat o studenten van de lerarenopleidingen o leraren in hun school

Technical note: Interpretation of field observations of point-source methane plume using observation-driven large-eddy simulations

This study demonstrates the ability of large-eddy simulation (LES) forced by a large-scale model to reproduce plume dispersion in an actual field campaign. Our aim is to bring together field observations taken under non-ideal conditions and LES to show that this combination can help to derive point-source strengths from sparse observations. We analyze results from a single-day case study based on data collected near an oil well during the ROMEO campaign (ROmanian Methane Emissions from Oil and gas) that took place in October 2019. We set up our LES using boundary conditions derived from the meteorological reanalysis ERA5 and released a point source in line with the configuration in the field. The weather conditions produced by the LES show close agreement with field observations, although the observed wind field showed complex features due to the absence of synoptic forcing. In order to align the plume direction with field observations, we created a second simulation experiment with manipulated wind fields that better resemble the observations. Using these LESs, the estimated source strengths agree well with the emitted artificial tracer gas plume, indicating the suitability of LES to infer source strengths from observations under complex conditions. To further harvest the added value of LES, higher-order statistical moments of the simulated plume were analyzed. Here, we found good agreement with plumes from previous LES and laboratory experiments in channel flows. We derived a length scale of plume mixing from the boundary layer height, the mean wind speed and convective velocity scale. It was demonstrated that this length scale represents the distance from the source at which the predominant plume behavior transfers from meandering dispersion to relative dispersion

Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH