DISSECTION OF THE MOLECULAR PATHWAYS INVOLVED IN PANCREATIC CANCER INITIATION AND PROGRESSION WITH A NOVEL IN VIVO APPROACH

We have seen great advances in our knowledge of the genetic regulation of various cancers in recent years, thanks in large part to large-scale genome sequencing efforts. As we catalogue and characterize the genomic aberrations associated with cancers with increasing detail and accuracy, we are faced with the challenge of having to cull bystanders from biologically active drivers and establish relevant disease context in which these drivers are rate-limiting. To address this challenge, we have adapted a loss-of-function screening approach to function in the context of an intact tumor microenvironment using patient-derived xenografts that more faithfully recapitulate the human disease compared to established cell lines. Due to the relevant genetic heterogeneity between human tumors with the same clinico-pathological indications, we have integrated independent screening approaches in a flexible platform for the interrogation of patient-derived samples as well as genetically-defined mouse models in exactly the same experimental conditions. The goal of this platform is to identify context-specific genetic vulnerabilities and translate these findings into drug discovery opportunities. As proof of concept for this approach, we describe the development of an in vivo loss-of-function screen to systematically interrogate epigenetic dependencies in pancreatic ductal adenocarcinoma (PDAC). In addition to the well-known genetic alterations (Kras, TP53, CDKN2A/p16, SMAD4), some epigenetic mechanisms demonstrated to play a central role in PDAC evolution and progression. The screening system utilizes tumor cells isolated from low-passage PDAC xenografted tissue and a lentiviral library of pooled shRNAs targeting 236 potentially \u201ddruggable\u201d epigenetic regulators. The custom-designed shRNA library (10 shRNAs per gene) was engineered with unique molecular barcodes that allow quantitation of each clone by massively parallel sequencing. Hairpins are clustered according to their depletion or enrichment in comparison to a control population before transplantation. To date, we have completed a total of 5 in vivo screens using diverse PDAC target cell models that have informed on novel epigenetic dependencies. So far, the main limitation for the systematic exploitation of in vivo loss-of-function screens to identify specific patient vulnerabilities come from the limited number of human cells contributing to tumor establishment in a transplantation setting. The frequency of these tumors initiating cells (TICs) is commonly estimated by time-consuming limiting dilution assays and may consistently vary between different tumor origins. With this in mind, we have integrated in our platform a system based on scrambled barcoded libraries that allows to directly assess the required coverage of screening libraries in each model and adjust the shRNA screens for this factor. Our coverage study demonstrated to be a powerful tool to identify the minimal number of cells/barcode required to sustain a complex library in transplantation assay and at the same time a step forward to personalize the in vivo screening patient by patient. We optimized a comprehensive data analytics pipeline and developed a high-throughput validation scheme to triage "hits" that emerge from each screen. The most potent "hits" have been enrolled in both functional and clinico-pathological validation studies to determine the highest priority targets for this devastating disease. Significantly, different components of the COMPASS histone H3 Lys4 (H3K4) methyltransferase complexes were identified as candidates in our screens. COMPASS and COMPASS-like complexes are characterized by unique subunits composition, whose identities provide insight into the different biological functions of these complexes. The methyltransferase unit of the COMPASS complexes is directly involved into the methylation of Lys4 on histone H3, a commonly accepted sign of open-chromatin and active transcription. Chromosomal translocations involving MLL gene are frequent events characterizing the Mixed Lineage Leukemia. In this disease, it has been shown that fusion events with a variety of different partners compromise the MLL methyltransferase activity. However, multiple members of the MLL family could be deregulated via different oncogenic mechanisms in PDAC, as the genetic alteration in MLL2 (amplification) and MLL3 (mutation) suggested. Our platform represents an ideal starting point to understand the COMPASS functionalities. So, a deeper understanding of genes and pathways regulated by each MLL subunit in the context of PDAC is critical to better elucidate the molecular dynamics of this disease and identify additional key points of vulnerability. Our study identified the core different subunits of the COMPASS complexes (WDR5-ASH2L-RBBP5) as broad relevant players in sustain PDAC progression, while the dependency on the MLL subunits appears to be more context-dependent and potentially consequent to specific genetic alterations. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, a critical ATR substrate, and c-Myc, also found to interact with WDR5. By showing that ATR inhibition mimicked the effects of WDR5 suppression, we open up the possibility of testing inhibitors currently in development for activity in this disease. These findings are proposing a new layer of complexities in trapping the COMPASS complexes during tumor development and unmasking unexplored directions for new therapeutical opportunities

Analysis of the diffusion process by pH indicator in microfluidic chips for liposome production

In recent years, the development of nano- and micro-particles has attracted considerable interest from researchers and enterprises, because of the potential utility of such particles as drug delivery vehicles. Amongst the different techniques employed for the production of nanoparticles, microfluidic-based methods have proven to be the most effective for controlling particle size and dispersity, and for achieving high encapsulation efficiency of bioactive compounds. In this study, we specifically focus on the production of liposomes, spherical vesicles formed by a lipid bilayer encapsulating an aqueous core. The formation of liposomes in microfluidic devices is often governed by diffusive mass transfer of chemical species at the liquid interface between a solvent (i.e., alcohol) and a non-solvent (i.e., water). In this work, we developed a new approach for the analysis of mixing processes within microfluidic devices. The method relies on the use of a pH indicator, and we demonstrate its utility by characterizing the transfer of ethanol and water within two different microfluidic architectures. Our approach represents an effective route to experimentally characterize diffusion and advection processes governing the formation of vesicular/micellar systems in microfluidics, and can also be employed to validate the results of numerical modelling

Quantifying ultrasonic deformation of cell membranes with ultra-high-speed imaging

We present a new method for controllable loading of cell models in an ultrasonic (20 kHz) regime. The protocol is based on the inertial-based ultrasonic shaking test and allows to deform cells in the range of few mm/m to help understand potential consequences of repeated loading characteristic of ultrasonic cutting

Tetra­aqua­bis(biuret-κ2 O,O′)yttrium(III) trichloride

In the title compound, [Y(C2H5N3O2)2(H2O)4]Cl3, the Y3+ ion (site symmetry 2) is bonded to eight O atoms (arising from two O,O′-bidentate biuret mol­ecules and four water mol­ecules) in a distorted square-anti­prismatic arrangement. A network of N—H⋯O, N—H⋯Cl and O—H⋯Cl hydrogen bonds help to establish the packing, leading to a three-dimensional network. One of the chloride ions has site symmetry 2

Detailed estimation of bioinformatics prediction reliability through the Fragmented Prediction Performance Plots

<p>Abstract</p> <p>Background</p> <p>An important and yet rather neglected question related to bioinformatics predictions is the estimation of the amount of data that is needed to allow reliable predictions. Bioinformatics predictions are usually validated through a series of figures of merit, like for example sensitivity and precision, and little attention is paid to the fact that their performance may depend on the amount of data used to make the predictions themselves.</p> <p>Results</p> <p>Here I describe a tool, named Fragmented Prediction Performance Plot (FPPP), which monitors the relationship between the prediction reliability and the amount of information underling the prediction themselves. Three examples of FPPPs are presented to illustrate their principal features. In one example, the reliability becomes independent, over a certain threshold, of the amount of data used to predict protein features and the intrinsic reliability of the predictor can be estimated. In the other two cases, on the contrary, the reliability strongly depends on the amount of data used to make the predictions and, thus, the intrinsic reliability of the two predictors cannot be determined. Only in the first example it is thus possible to fully quantify the prediction performance.</p> <p>Conclusion</p> <p>It is thus highly advisable to use FPPPs to determine the performance of any new bioinformatics prediction protocol, in order to fully quantify its prediction power and to allow comparisons between two or more predictors based on different types of data.</p

The accumulation of particles in ureteric stents is mediated by flow dynamics: Full-scale computational and experimental modeling of the occluded and unoccluded ureter

Ureteric stents are clinically deployed to restore urinary drainage in the presence of ureteric occlusions. They consist of a hollow tube with multiple side-holes that enhance urinary drainage. The stent surface is often subject to encrustation (induced by crystals-forming bacteria such as Proteus mirabilis) or particle accumulation, which may compromise stent's drainage performance. Limited research has, however, been conducted to evaluate the relationship between flow dynamics and accumulation of crystals in stents. Here, we employed a full-scale architecture of the urinary system to computationally investigate the flow performance of a ureteric stent and experimentally determine the level of particle accumulation over the stent surface. Particular attention was given to side-holes, as they play a pivotal role in enhancing urinary drainage. Results demonstrated that there exists an inverse correlation between wall shear stress (WSS) and crystal accumulation at side-holes. Specifically, side-holes with greater WSS levels were those characterized by inter-compartmental fluid exchange between the stent and ureter. These "active " side-holes were located either nearby ureteric obstructions or at regions characterized by a physiological constriction of the ureter. Results also revealed that the majority of side-holes (> 60%) suffer from low WSS levels and are, thus, prone to crystals accumulation. Moreover, side-holes located toward the proximal region of the ureter presented lower WSS levels compared to more distal ones, thus suffering from greater particle accumulation. Overall, findings corroborate the role of WSS in modulating the localization and extent of particle accumulation in ureteric stents. (C) 2022 Author(s)

Effects of Isolated Systolic Hypertension and Essential Hypertension on Large and Middle-sized Artery Compliance

Systolic hypertension of the elderly is characterized by a reduction in arterial compliance. Whether and to what extent this involves arteries of various structure and size is not well known.To study carotid and radial artery compliance in systolic hypertension of the elderly, compared to essential hypertension and normotension.We investigated 28 elderly patients with systolic hypertension (age 68.6 +/- 1.4 years, mean +/- SE; systolic blood pressure160 mmHg and diastolic blood pressure90 mmHg) plus 17 age-matched patients with essential hypertension and 15 age-matched healthy normotensive subjects. Radial and carotid artery compliance were evaluated using echotracking techniques. In both arteries compliance was assessed statistically and dynamically, i.e. as compliance values throughout the diasto-systolic pressure range. Measurements included intima-media wall thickness of the radial artery.Compared to normotensive subjects, carotid artery compliance was reduced in essential hypertension and more so in systolic hypertension. However, although in both groups radial artery wall thickness was markedly greater than in the normotensive group, radial artery compliance was markedly reduced in systolic hypertension, but unchanged in essential hypertension.In systolic hypertension of the elderly the reduction of arterial compliance is marked in both muscular and large elastic arteries, while in elderly essential hypertensives changes in arterial compliance are more heterogeneous, i.e. only carotid artery compliance is reduced. The different effects of these two types of hypertension on arterial mechanics are visible throughout the physiological range of blood pressure and probably accounted for by different alterations in vessel wall structure

Foam-in-Vein: Rheological Characterisation of Liquid Sclerosing Foams Using a Pipe Viscometer

Sclerotherapy is one of the most common and least-invasive treatment methods for varicose veins. While bench-top properties of sclerosing foams (e.g., bubble size distribution and foam half-life) have been studied previously, their flow behaviour and its relationship to therapeutic efficacy remain largely uncharacterised. To address this research gap, the present study reports on a novel approach for the rheological characterisation of sclerosing foams aimed at obtaining clinically-applicable data. A pipe viscometry apparatus was employed under conditions that mimic the end-point therapeutic application of foams. Polidocanol (1% v/v) foams of various liquid-to-gas volume ratios (1:3, 1:4 and 1:5) were formulated manually using the Tessari and DSS (double syringe system) methods across a clinically-relevant range of shear rates (≈ 7 s^{-1} – 400 s^{-1}), in polytetrafluoroethylene pipes of different diameters (2.48 mm and 4.48 mm). Additionally, end-effect and wall-slip correction methods were utilised to model the nominal rheology of sclerosing foams. The rheological data were fitted into a power-law model to obtain fluid flow index (n) and fluid consistency index (K) of sclerosing foams, followed by an in-depth statistical analysis of the power-law indices. The observed rheological behaviour of sclerosing foams is shown to be dependent on vessel diameter and liquid-to-gas ratio, while the type of manual formulation technique used appears to be statistically insignificant towards foam rheology. Sclerosing foams behaved as shear-thinning fluids with observed flow indices ranging 0.238 < n < 0.445, while the observed consistency indices ranged 2.977 < K < 12.49. The nominal (end-effect corrected) rheology of foams was shown to follow similar trends concerning liquid-to-gas ratio and formulation technique, independent of the tube diameter. The power-law characterisation of sclerosing foam rheology provided evidence of a quasi-static drainage effect that affected foam viscosity during slower injections. Wall-slip correction failed to provide physically meaningful results and statistical analysis suggested that the type of manual formulation technique used has no impact on the outcome of sclerotherapy on larger varicosities. Overall, results suggest a direct correlation between foam dryness and viscosity. Based on the developed rheological model, this work also demonstrates that low injection flowrates could yield higher therapeutic efficacy in dilated varicose veins