13 research outputs found
Divergent Innate and Epithelial Functions of the RNA-Binding Protein HuR in Intestinal Inflammation
HuR is an abundant RNA-binding protein acting as a post-transcriptional regulator of many RNAs including mRNAs encoding inflammatory mediators, cytokines, death signalers and cell cycle regulators. In the context of intestinal pathologies, elevated HuR is considered to enhance the stability and the translation of pro-tumorigenic mRNAs providing the rationale for its pharmacological targeting. However, HuR also possesses specific regulatory functions for innate immunity and cytokine mRNA control which can oppose intestinal inflammation and tumor promotion. Here, we aim to identify contexts of intestinal inflammation where the innate immune and the epithelial functions of HuR converge or diverge. To address this, we use a disease-oriented phenotypic approach using mice lacking HuR either in intestinal epithelia or myeloid-derived immune compartments. These mice were compared for their responses to (a) Chemically induced Colitis; (b) Colitis- associated Cancer (CAC); (c) T-cell mediated enterotoxicity; (d) Citrobacter rodentium-induced colitis; and (e) TNF-driven inflammatory bowel disease. Convergent functions of epithelial and myeloid HuR included their requirement for suppressing inflammation in chemically induced colitis and their redundancies in chronic TNF-driven IBD and microbiota control. In the other contexts however, their functions diversified. Epithelial HuR was required to protect the epithelial barrier from acute inflammatory or infectious degeneration but also to promote tumor growth. In contrast, myeloid HuR was required to suppress the beneficial inflammation for pathogen clearance and tumor suppression. This cellular dichotomy in HuR's functions was validated further in mice engineered to express ubiquitously higher levels of HuR which displayed diminished pathologic and beneficial inflammatory responses, resistance to epithelial damage yet a heightened susceptibility to CAC. Our study demonstrates that epithelial and myeloid HuR affect different cellular dynamics in the intestine that need to be carefully considered for its pharmacological exploitation and points toward potential windows for harnessing HuR functions in intestinal inflammation
Detection of Pathogenic Mycobacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation
Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 104 bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples
The RNA binding protein HuR is a gatekeeper of liver homeostasis
BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is initiated by steatosis and can progress via fibrosis and cirrhosis to hepatocellular carcinoma (HCC). The RNA binding protein HuR controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte-HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or a NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis and HCC development were studied by histology, flow cytometry, quantitative PCR and RNA sequencing. The liver lipidome was characterized by lipidomics analysis and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation-sequencing. Hepatocyte-specific HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition, compared to control HuR-sufficient mice. On a NAFLD-inducing diet, hepatocyte-specific HuR-deficiency resulted in exacerbated inflammation, fibrosis and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics and RNA-immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady-state, a triglyceride signature resembling that of NAFLD livers. Moreover, upregulation of Spp1 and its product osteopontin mediated, at least partially, the fibrosis development in hepatocyte-specific HuR deficiency on a NAFLD-inducing diet, as shown by experiments utilizing antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically
Detection of Leishmania spp. using nanoparticles
Leishmaniosis is a parasitic zoonotic disease that represents a significant publichealth threat. The accurate diagnosis of the disease is of determinative importance inthe application of targeted treatment and minimizes the danger of transmissioncontributing to the decrease of the number of cases and the limitation of theirgeographic distribution. The use of bio-nanoparticles could contribute significantly tothe detection of the pathogen in clinical samples without the need of dedicatedequipment.The aim of this study was the design of an easily applicable, fast, specific andreliable technique for the detection of Leishmania spp. using cadmium selenide(CdSe) quantum dots and gold nanoparticles.Initially, a comparative evaluation of four PCR methods was performed for thedetection of Leishmania spp. in order to define the optimum target-DNA region of theLeishmania genome, and conclude to a reference methodology. Four (4)oligonucleotide-probes were designed to be conjugated with commercially availablegold nanoparticles for the detection of Leishmania DNA. For the same reason(detection of Leishmania DNA) two oligonucleotide-probes were designed, the one tobe conjugated with quantum dots and the other with magnetic beads. For the detectionof surface antigen of Leishmania, quantum dots and magnetic beads were conjugatedwith two antibodies specific for parasite’s surface antigens. The evaluation of themethods under study was performed in regard to their sensitivity, specificity, minimum detection limit (MDL), and specifically for the PCR methods, repeatability and reproducibility. The reference material used consisted of culturedisolates and clinical samples that were collected from dogs brought to veterinaryclinics with suspicion of leismaniosis. Similar results were recorded by all PCR methods with regard mainly to culturedisolates. The percentage of positive results recorded by method C was significantlyhigher compared to the other three (58.1%). The respective measurement for methodsA, B, and D, was similar and varied in all cases between 19 and 25%. The relativesensitivity and specificity were referenced to method C that produced the highestpercentage of confirmed positive and negative results. Effectively sensitivity andspecificity of methods A, B, and D were defined to 50.7% (33/65), 43% (28/65), 40%(26/65) and 90.8% (69/76), 93.4% (71/76), 89.5% (68/76) respectively. TheMDL of the methods A-D was calculated as the mean average of the measurement ofseven repeats, at 30.7, 5, 3.7, and 5 promastigotes/ml, respectively. Repeatabilitywas excellent for the all methods, as it was higher than 0.75, something that was statistically significant at 5% level. Reproducibility was good to excellent dependingon the PCR reagents that were used. The positive and the negative controls werecorrectly identified with all three combinations of nanoparticles. The relativesensitivity and specificity of the DNA detection method with gold nanoparticlesreferenced to PCR, were 92% and100% respectively depending on the material to beexamined, while the MDL was defined at 11,5ng/ml. The MDL of the DNA detectionmethod using quantum dots was 3.125ng/ml, while the specificity of cellular detectionmethod with quantum dots was 103 cells/ml.The three methods described in the present study provide an easy, fast andeconomic way for the detection of members of genus Leishmania using nano-probes.The proposed methods can be used for the development of diagnostic methodsfor use particularly in enzootic regions where detection of very small amounts ofparasite is not of top priority.Η λεϊσμανίωση είναι μια παρασιτική νόσος του σκύλου που αποτελεί σημαντικόπρόβλημα για τη δημόσια υγεία. Η έγκαιρη και ακριβής διάγνωση της ασθένειας είναικαθοριστικής σημασίας στην εφαρμογή στοχευμένης θεραπείας και μειώνει τονκίνδυνο μετάδοσης, συμβάλλοντας στον περιορισμό του αριθμού των κρουσμάτωνκαι της γεωγραφικής εξάπλωσης. Η χρήση βιονανοσυστημάτων θα μπορούσε νασυνεισφέρει σημαντικά στη διευκόλυνση της ανίχνευσης του συγκεκριμένουπαθογόνου σε κλινικά δείγματα χωρίς τη χρήση εξειδικευμένων συσκευών υψηλούκόστους.Σκοπός της παρούσας μελέτης είναι να αναπτυχθεί μια εύκολα εφαρμόσιμη,γρήγορη, ειδική και αξιόπιστη μέθοδος ανίχνευσης της Leishmania spp. χρησιμοποιώντας κβαντικά κοκκία σεληνιούχου καδμίου και νανοσωματίδια χρυσού.Αρχικά, έγινε συγκριτική αξιολόγηση τεσσάρων μεθόδων PCR για ανίχνευση τηςLeishmania spp. προκειμένου να επιλεχθεί η κατάλληλη περιοχή-στόχος τουγενώματος της Leishmania που θα στόχευαν οι ολιγονουκλεοτιδικοί-ανιχνευτές καινα αναδειχθεί μια επαρκής μέθοδος αναφοράς. Σχεδιάστηκαν στη συνέχεια 4ολιγονουκλεοτιδικοί-ανιχνευτές που συνδέθηκαν με εμπορικά διαθέσιμανανοσωματίδια χρυσού για την ανίχνευση του DNA της Leishmania. Για τον ίδιοσκοπό (ανίχνευση DNA της Leishmania) σχεδιάστηκαν δύο ολιγονουκλεοτιδικοί- ανιχνευτές, ένας για σύνδεση με κβαντικά κοκκία και ο άλλος για σύνδεση μεμαγνητικά σφαιρίδια. Για την ανίχνευση ενός επιφανειακού αντιγόνου τηςLeishmania, τα κβαντικά κοκκία και τα μαγνητικά σφαιρίδια συνδέθηκαν με δύοειδικά για το παράσιτο αντισώματα. Η αξιολόγηση των υπό μελέτη μεθόδων έγινεαναφορικά με την ευαισθησία τους, την ειδικότητά τους, το ελάχιστο όριο ανίχνευσήςτους (Ε.Ο.Α.), και ειδικά για τις μεθόδους PCR, την επαναληψιμότητα και τηναναπαραγωγιμότητά τους. Το υλικό αναφοράς που χρησιμοποιήθηκε συνίστατο απόστελέχη απομονωμένα σε καλλιέργεια και κλινικά δείγματα που συλλέχθηκαν απόσκύλους που είχαν προσκομιστεί σε κτηνιατρικές κλινικές με υποψία λεϊσμανίωσης.Με όλες τις μεθόδους PCR που εξετάστηκαν καταγράφηκαν παρόμοιααποτελέσματα κυρίως αναφορικά με τα καλλιεργημένα στελέχη. Το υψηλότεροποσοστό θετικών δειγμάτων ανιχνεύτηκε με τη μέθοδο Γ (58.1%). Με τις άλλες τρειςμεθόδους PCR, το ποσοστό θετικότητας ήταν σημαντικά χαμηλότερο και κυμάνθηκεσε όλες τις περιπτώσεις μεταξύ 19 και 25%. Η σχετική ευαισθησία και ειδικότηταεκτιμήθηκε αναφορικά με τη μέθοδο Γ, με την οποία ανιχνεύτηκε το υψηλότεροποσοστό επιβεβαιωμένων θετικών και αρνητικών αποτελεσμάτων. Έτσι για τιςμεθόδους Α, Β, και Δ, η ευαισθησία και ειδικότητα διαμορφώθηκε αντίστοιχα στο 50.7% (33/65), 43% (28/65), 40% (26/65) και 90.8% (69/76), 93.4% (71/76) 89.5%(68/76). Το μέσο ελάχιστο όριο ανίχνευσης των μεθόδων Α-Δ σε επτά επαναλήψειςυπολογίστηκε αντίστοιχα στα 30.7, 5, 3.7, και 5 προμαστιγωτά/ml. Ηεπαναληψιμότητα ήταν εξαιρετική για όλες τις μεθόδους αφού ήταν υψηλότερη από0.75, κάτι που ήταν στατιστικά σημαντικό σε επίπεδο 5%, ενώ η αναπαραγωγιμότηταήταν καλή έως εξαιρετική ανάλογα με τα αντιδραστήρια PCR πουχρησιμοποιήθηκαν. Οι θετικοί και οι αρνητικοί μάρτυρες ανιχνεύτηκαν σωστά και μετους τρεις συνδυασμούς νανοσωματιδίων. Η σχετική ευαισθησία και ειδικότητα τηςμεθόδου ανίχνευσης DNA με τα νανοσωματίδια χρυσού συγκριτικά με τη μέθοδοςαναφοράς, προσδιορίστηκε σε 92% και 100% αντίστοιχα αναλόγως του εξεταζόμενουυλικού, ενώ το Ε.Ο.Α., σε 11,5 ng/μl. Οι αντίστοιχες τιμές που καταγράφηκαν στοπλαίσιο του προσδιορισμού της ειδικότητας και της ευαισθησίας της μεθόδουανίχνευσης DNA με χρήση κβαντικών κοκκίων ήταν 3,125 ng/μl, ενώ η ειδικότητατης μεθόδου ανίχνευσης των κυττάρων του πρωτοζώου με κβαντικά κοκκία ήταν 103κύτταρα/ml. Οι τρεις μέθοδοι που περιγράφηκαν στην παρούσα μελέτη παρέχουν ένανεύκολο, γρήγορο και οικονομικό τρόπο για την ανίχνευση μελών του γένουςLeishmania με τη χρήση των συγκεκριμένου τύπου νανο-ανιχνευτών. Οιπροτεινόμενες μέθοδοι μπορεί να αποτελέσουν βάση για την ανάπτυξη διαγνωστικώνμεθόδων ελέγχου για χρήση σε ενζωοτικές περιοχές ιδίως εάν λόγω διαθέσιμωνπόρων δεν αποτελεί προτεραιότητα η ανίχνευση πολύ μικρής ποσότητας τουπαρασίτου
Specific detection of unamplified mycobacterial DNA using fluorescent semiconductor quantum dots and magnetic beads
Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp. dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmim selenite QDs conjugated with streptavidin and species specific probes were used to produce fluorescent signal. MBs conjugated with streptavidin and a genus specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method on isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined at 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage of patients with tuberculosis, and Mycobacterium avium subsp. paratuberculosis in faeces and paraffin embedded tissues, in comparison with culture, Ziehl-Neelsen stain and Real Time PCR. The concordance of these methods compared to the proposed with connection to positive and negative samples varied between 53.84% - 87.23% and 84.61%-100% respectively. The overall accuracy of the QD assay compared to Real Time PCR was 70-90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples
Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles
Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples
MBs/bacteria/QDs complexes visualized by fluorescent microscopy.
<p>Magnetic beads (arrows) are surrounded by a fluorescent halo with shape and dimensions compatible with mycobacteria. 1000X.</p
Relative fluorescence values of BCG and <i>E. coli</i> serial dilutions.
<p>Relative fluorescence values of BCG and <i>E. coli</i> serial dilutions.</p
Bacterial cells (a) are separated from the matrix using MBs coupled with genus specific polyclonal antibodies (b) and a magnetic device (c).
<p>These complexes are then tagged with anti-HBHA (d) and anti-mouse biotynylated antibody and streptavidin-conjugated QDs (e) which lead to the detection of a fluorescent signal (f).</p
The scaffold protein IQGAP1 links heat-induced stress signals to alternative splicing regulation in gastric cancer cells
Data de publicació electrònica: 23-07-2021In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation.InfrafrontierGR/Phenotypos Infrastructure, co-funded by Greece and the European Union (European Regional Development Fund) [NSRF 2014–2020, MIS 5002135]; Hellenic Foundation for Research & Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT) [grant agreement 846 to ZE]; MR was supported by the European Research Council [ERC AdvG 670146]; European Commission Grant FP7-PEOPLE-2010-IEF [274837] to PK; Stavros Niarchos Foundation (SNF) donation to BSRC “Al. Fleming”