Μελέτη αλληλεπίδρασης συνθετικών πολυηλεκτρολυτών με το ένζυμο Φωσφορυλάση του Γλυκογόνου σε υδατικά διαλύματα

Η διερεύνηση των αλληλεπιδράσεων μεταξύ πολυηλεκτρολυτών και πρωτεϊνών παραμένει ένας από τους πιο σημαντικούς ερευνητικούς τομείς στη βιοτεχνολογία. Είναι ευρέως γνωστό πως οι πρωτεΐνες και οι πολυηλεκτρολύτες μπορούν να σχηματίσουν δομές καθοδηγούμενες από μη ειδικές και μη-ομοιοπολικές αλληλεπιδράσεις, κυρίως ηλεκτροστατικές αλληλεπιδράσεις και αλληλεπιδράσεις Van der Waals. H Φωσφορυλάση του Γλυκογόνου (GP) είναι ένα αλλοστερικό ένζυμο, το οποίο αποτελεί κλειδί στον μεταβολισμό του γλυκογόνου ρυθμίζοντας τον ρυθμό αποικοδόμησης αυτού και επομένως των επιπέδων της γλυκόζης στην κυκλοφορία του αίματος. Για τον παραπάνω λόγο αξιοποιείται ως μοριακός στόχος για τον κατευθυνόμενο από τη δομή σχεδιασμό δυνάμει αντιδιαβητικών φαρμάκων. Τα φυσικοχημικά και βιοχημικά χαρακτηριστικά τόσο του πολυμεθακρυλικού εστέρα της 2-διμεθυλάμινο αιθυλικής αλκοόλης (poly 2-(dimethylamino)ethyl methacrylate, PDMAEMA), που είναι ένα pH αποκρινόμενο, καλά χαρακτηρισμένο πολυμερές, βιοσυμβατό με ιδιαίτερα χαμηλά επίπεδα τοξικότητας, όσο και του κατιοντικού αναλόγου του τεταρτοταγούς ιωδιούχου αμμωνιακού άλατος (quaternized poly 2-(dimethylamino)ethyl methacrylate, QPDMAEMA), το οποίο παρουσιάζει θετικά φορτία κατά μήκος της πολυμερικής αλυσίδας, μεγάλη διαλυτότητα και αποκρισιμότητα στη μεταβολή της ιοντικής ισχύος του διαλύματος, θεωρήθηκαν κατάλληλα για τη χρήση των ως φορέων αλληλεπίδρασης με την GP. Στο πλαίσιο της παρούσας πτυχιακής εργασίας πραγματοποιήθηκαν φυσικοχημικές μελέτες χαρακτηρισμού πολυμερών και βιομορίων, διερευνώντας τόσο την αλληλεπίδραση της GP με τους εν λόγω πολυηλεκτρολύτες όσο και την μορφολογία και τις ιδιότητες των σχηματιζόμενων μικτών νανοδομών.The interactions between polymers and proteins remain one of the most important research fields in biotechnology. It is widely known that proteins and polyelectrolytes can develop structures guided by non-convalent interactions. The interactions that take place are electrostatic and Van der Waals interactions. Glycogen Phosphorylase (GP) is an allosteric enzyme which is particularly important in glycogen metabolism by regulating the rate of glycogen phosphorolysis and consequently the circulating glucose levels. For the aforementioned reasons this enzyme has been utilized as a molecular target for structure-based antidiabetic drug design. The physicochemical and biophysical properties of poly(2-(dimethylamino)ethyl methacrylate), PDMAEMA which is a pH responsive, very well characterized, biocompatible with especially low toxicity levels and the cationic analogue of the quaternized ammonium iodide salt (quaternized poly 2-(dimethylamino) ethyl methacrylate, QPDMAEMA) which has positive charges along the polymer chain, high solubility in aqueous solutions and responsiveness to changes in the ionic strength of the solution, were considered suitable for use as GP interaction carriers. In the context of the present thesis, physicochemical characterization studies of polymers and biomolecules were performed, investigating both the interaction of GP with polyelectrolytes and the morphology and properties of the formed mixed nanostructures

Deep learning for whole slide image analysis : an overview

The widespread adoption of whole slide imaging has increased the demand for effective and efficient gigapixel image analysis. Deep learning is at the forefront of computer vision, showcasing significant improvements over previous methodologies on visual understanding. However, whole slide images have billions of pixels and suffer from high morphological heterogeneity as well as from different types of artifacts. Collectively, these impede the conventional use of deep learning. For the clinical translation of deep learning solutions to become a reality, these challenges need to be addressed. In this paper, we review work on the interdisciplinary attempt of training deep neural networks using whole slide images, and highlight the different ideas underlying these methodologies.Publisher PDFPeer reviewe

Assessment of immunological features in muscle-invasive bladder cancer prognosis using ensemble learning

Funding: This research received financial support from Definiens GmbH and the Industrial Centre for AI Research in digital Diagnostics (iCAIRD) which is funded by Innovate UK on behalf of UK Research and Innovation (UKRI) [project number: 104690].The clinical staging and prognosis of muscle-invasive bladder cancer (MIBC) routinely includes the assessment of patient tissue samples by a pathologist. Recent studies corroborate the importance of image analysis in identifying and quantifying immunological markers from tissue samples that can provide further insight into patient prognosis. In this paper, we apply multiplex immunofluorescence to MIBC tissue sections to capture whole-slide images and quantify potential prognostic markers related to lymphocytes, macrophages, tumour buds, and PD-L1. We propose a machine-learning-based approach for the prediction of 5 year prognosis with different combinations of image, clinical, and spatial features. An ensemble model comprising several functionally different models successfully stratifies MIBC patients into two risk groups with high statistical significance (p value < 1×10−5). Critical to improving MIBC survival rates, our method correctly classifies 71.4% of the patients who succumb to MIBC, which is significantly more than the 28.6% of the current clinical gold standard, the TNM staging system.Publisher PDFPeer reviewe

Bacteremia During the First Year After Solid Organ Transplantation: An Epidemiological Update.

BACKGROUND There are limited contemporary data on the epidemiology and outcomes of bacteremia in solid organ transplant recipients (SOTr). METHODS Using the Swiss Transplant Cohort Study registry from 2008 to 2019, we performed a retrospective nested multicenter cohort study to describe the epidemiology of bacteremia in SOTr during the first year post-transplant. RESULTS Of 4383 patients, 415 (9.5%) with 557 cases of bacteremia due to 627 pathogens were identified. One-year incidence was 9.5%, 12.8%, 11.4%, 9.8%, 8.3%, and 5.9% for all, heart, liver, lung, kidney, and kidney-pancreas SOTr, respectively (P = .003). Incidence decreased during the study period (hazard ratio, 0.66; P < .001). One-year incidence due to gram-negative bacilli (GNB), gram-positive cocci (GPC), and gram-positive bacilli (GPB) was 5.62%, 2.81%, and 0.23%, respectively. Seven (of 28, 25%) Staphylococcus aureus isolates were methicillin-resistant, 2/67 (3%) enterococci were vancomycin-resistant, and 32/250 (12.8%) GNB produced extended-spectrum beta-lactamases. Risk factors for bacteremia within 1 year post-transplant included age, diabetes, cardiopulmonary diseases, surgical/medical post-transplant complications, rejection, and fungal infections. Predictors for bacteremia during the first 30 days post-transplant included surgical post-transplant complications, rejection, deceased donor, and liver and lung transplantation. Transplantation in 2014-2019, CMV donor-negative/recipient-negative serology, and cotrimoxazole Pneumocystis prophylaxis were protective against bacteremia. Thirty-day mortality in SOTr with bacteremia was 3% and did not differ by SOT type. CONCLUSIONS Almost 1/10 SOTr may develop bacteremia during the first year post-transplant associated with low mortality. Lower bacteremia rates have been observed since 2014 and in patients receiving cotrimoxazole prophylaxis. Variabilities in incidence, timing, and pathogen of bacteremia across different SOT types may be used to tailor prophylactic and clinical approaches

Do Infectious Diseases After Kidney Retransplantation Differ From Those After First Kidney Transplantation?

Background Infectious diseases (IDs) are highly relevant after solid organ transplantation in terms of morbidity and mortality, being among the most common causes of death. Patients undergoing kidney retransplantation (re-K-Tx) have been already receiving immunosuppressive therapy over a prolonged period, potentially facilitating subsequent infections. Comparing ID events after re-K-Tx and first kidney transplantation (f-K-Tx) can delineate patterns and risks of ID events associated with prolonged immunosuppression. Methods We included adult patients with records on f-K-Tx and re-K-Tx in the Swiss Transplant Cohort Study. We analyzed ID events after f-K-Tx and re-K-Tx within the same patients and compared infection rates, causative pathogens, and infection sites. Recurrent time-to-event analyses were performed for comparison of infection rates. Results A total of 59 patients with a median age of 47 years (range, 18–73) were included. Overall, 312 ID events in 52 patients occurred. In multivariable recurrent event modeling, the rate of ID events was significantly lower after re-K-Tx (hazard ratio, 0.70; P = .02). More bacterial (68.9% vs 60.4%) and fungal (4.0% vs 1.1%) infections were observed after f-K-Tx but fewer viral infections (27.0% vs 38.5%) as compared with re-K-Tx (P = .11). After f-K-Tx, urinary and gastrointestinal tract infections were more frequent; after re-K-Tx, respiratory tract and surgical site infections were more frequent (P &lt; .001). Conclusions ID events were less frequent after re-K-Tx. Affected sites differed significantly after f-K-Tx vs re-K-Tx

Peroral Amphotericin B Polymer Nanoparticles Lead to Comparable or Superior In Vivo Antifungal Activity to That of Intravenous Ambisome® or Fungizone™

Background: Despite advances in the treatment, the morbidity and mortality rate associated with invasive aspergillosis remains unacceptably high (70–90%) in immunocompromised patients. Amphotericin B (AMB), a polyene antibiotic with broad spectrum antifungal activity appears to be a choice of treatment but is available only as an intravenous formulation; development of an oral formulation would be beneficial as well as economical. Methodology: Poly(lactide-co-glycolode) (PLGA) nanoparticles encapsulating AMB (AMB-NPs) were developed for oral administration. The AMB-NPs were 113±20 nm in size with ~70% entrapment efficiency at 30% AMB w/w of polymer. The in vivo therapeutic efficacy of oral AMB-NPs was evaluated in neutropenic murine models of disseminated and invasive pulmonary aspergillosis. AMB-NPs exhibited comparable or superior efficacy to that of Ambisome® or Fungizone™ administered parenterally indicating potential of NPs as carrier for oral delivery. Conclusions: The present investigation describes an efficient way of producing AMB-NPs with higher AMB pay-load and entrapment efficiency employing DMSO as solvent and ethanol as non-solvent. The developed oral formulation was highly efficacious in murine models of disseminated aspergillosis as well as an invasive pulmonary aspergillosis, which is refractory to treatment with IP Fungizone™and responds only modestly to AmBisome®

Comparison of Human Memory CD8 T Cell Responses to Adenoviral Early and Late Proteins in Peripheral Blood and Lymphoid Tissue

Treatment of invasive adenovirus (Ad) disease in hematopoietic stem cell transplant (SCT) recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs) targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977) and late protein hexon (H-892) were compared in peripheral blood (PB) and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16–24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients

Gymnemic acids inhibit hyphal growth and virulence in Candida albicans

Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus