Crystal nucleation from solution: design and modelling of detection time experiments

Crystal nucleation is the process responsible for the appearance of a thermodynamically stable phase from a metastable parent solution. Given its activated nature, nucleation is affected by stochasticity which, despite originating at the molecular level, affects heavily also the macroscopic behaviour of the system. Being far too small to be observed directly, nuclei are detected by indirect methods, which correlate the formation of the new phase with a measurable change in a property of the system, hence a model linking nuclei formation and crystals detection is always needed. We have previously presented a model describing nucleation in macroscopic systems as a stochastic Poisson process. The model, despite its general character, can describe industrially relevant processes, e.g. batch cooling at different operating conditions. The different scales influenced by the stochastic nature of nucleation demand appropriate theoretical and experimental investigations, particularly for applying the model to industrial scale-up, optimisation, and control. Using statistical tools, we have looked into the issue of estimating stochastic processes by collecting a representative, but limited number of data, produced from a homogeneous set. Moreover, using our model, we analysed the sensitivity of crystallising systems on initial and boundary conditions, with particular emphasis on the effect of supersaturation, temperature and detection conditions. Finally, in light of the stochastic nature of nucleation, we also applied statistical meta-analysis to assess the agreement between the fitting and its parameters and experiments, to gain further insight into the quality of the model. Experimentally, we have first investigated the conditions to perform homogeneous and reproducible measurements, necessary to understand the fundamental physical features and ultimately to estimate reliable kinetic parameters. A second aspect we have explored concerned the size of the crystallising systems. Since in macroscopic reactors various phenomena occur simultaneously (nucleation, growth, breakage, agglomeration) we chose to work with two main system sizes, 1-3 mL reactors (mesoscale) and 1-60 nL reactors (microscale, i.e. microscopic droplets), where at least some of such phenomena could be decoupled. In the mesoscale crystallisers, one can perform experiments where temperature and transmissivity could be measured online, hence monitoring the appearance and disappearance of crystals. Additionally, the influence of fluid-dynamics, typically turbulent in these reactors, was investigated. In the microfluidic chips, on the other hand, a very high through-put (thousands of replicas of the same reactor) can be potentially achieved and, thanks to their very small size, high supersaturations, outside of usual experimental reach, could be explored. Additionally, within the microscopic droplets the fluid motion is generally diffusive or laminar convective, hence hindering breakage and agglomeration. One could thus observe systems where nucleation and growth of single crystals (or of few crystals) occur unperturbed. Nevertheless, some main challenges, which we have been addressing, must be tackled before performing reliable crystallisation experiments: the characterisation and the reproducibility of shape and size of the droplets and their stability (i.e. the loss of mass due to evaporation and perspiration through the chip). In conclusion, we demonstrate that, even if the data are reproducible and reliable, robust probability estimations can be obtained only with a sufficiently large number of experiments, which require careful design to avoid sensitivity regions and data processing to reject the non-homogeneous data. The different sizes investigated have permitted to gain a better insight into the fundamental phenomena occurring in a crystallising system between the first formation of nuclei until crystal detection, which is of utmost importance for understanding the design of the experiments at an industrially relevant scale. Moreover, appropriate mathematical tools allowed to assess the reliability of the fitting obtained from independent measurements of the same system at different conditions

Liver transplantation for hepatocellular carcinoma in a patient with a novel telomerase mutation and steatosis

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Light-Triggered Trafficking to the Cell Nucleus of a Cationic Polyamidoamine Functionalized with Ruthenium Complexes

Strategies for endosomal escape and access to the cell nucleus are highly sought for nanocarriers to deliver their load efficiently following endocytosis. In this work, we have studied the uptake and intracellular trafficking of a polycationic polyamidoamine endowed with a luminescent Ru complex, Ru-PhenAN, that shows unique trafficking to the cell nucleus. Live cell imaging confirmed the capacity of this polymer to access the nucleus, excluding artefacts due to cell fixation, and clarified that the mechanism of escape is light-triggered and relies on the presence of the Ru complexes and their capacity to absorb light and act as photosensitizers for singlet oxygen production. These results open up the possibility to use polyamidoamineruthenium complexes for targeted light-triggered delivery of genetic material or drugs to the cytosol and nucleus

Service learning, well-being and school performance : causal evidence from Italian High school students

Service learning (SL) is a relatively common pedagogical method in the US, where it has been widely adopted in schools, colleges, and universities. The method requires students to take part in the activities of organizations that serve community needs. While the literature argues that SL activities could generate beneficial effects for students' cognitive abilities, self-esteem and motivation, satisfaction with schools, attitudes towards institutions, and civic engagement, empirical evaluation of these effects is scarce and frequently far from rigorous. This paper investigates the effects of being engaged in “SL-like” activities on the school performance of 9th and 10th grade students at high risk of school failure and drop-out in Italy. We contribute to the empirical literature on SL in three ways. First, we run the first pilot randomized controlled trial (RCT) to simultaneously evaluate the effect of a SL program on both cognitive and non-cognitive skills, with the latter measured through questionnaires and incentivized tasks. Second, this is the first attempt to evaluate the impact of SL as a remedial intervention on the specific target of low-achieving students at risk of dropping out from school. Finally, this is the first attempt to rigorously investigate SL activities in Italy, and one of the first in Europe, as most RCTs involving SL have focused on the US. Our experimental results show that—consistently with the literature developed in the US—participation in “SL-like” activities leads to a general improvement in non-cognitive skills of students at risk of dropping out. On the other hand, contrary to what the literature argues, the intervention does not improve cognitive skills, since participation in the program even increases the risk of school failure. These results suggest designing and implementing SL interventions in schools with great care to avoid unintended negative consequences

The multi-handler knapsack problem under uncertainty

The Multi-Handler Knapsack Problem under Uncertainty is a new stochastic knapsack problem where, given a set of items, characterized by volume and random profit, and a set of potential handlers, we want to find a subset of items which maximizes the expected total profit. The item profit is given by the sum of a deterministic profit plus a stochastic profit due to the random handling costs of the handlers. On the contrary of other stochastic problems in the literature, the probability distribution of the stochastic profit is unknown. By using the asymptotic theory of extreme values, a deterministic approximation for the stochastic problem is derived. The accuracy of such a deterministic approximation is tested against the two-stage with fixed recourse formulation of the problem. Very promising results are obtained on a large set of instances in negligible computing time

Co-culture of Caco2 and HT-29 cells as an innovative method to mimic in vitro the morphology and permeability properties of human intestinal epithelium

For investigating the complexity of the human intestinal epithelium, a valid experimental approach is represented by co-culture. In the present study an intestinal co-culture Caco2/HT-29 (70/30) was set up starting from the parental populations of differentiated cells as previously described [1, 2]. Co-culture was harvested at 0 (T0), 6 (T6), and 14 (T14) days of post confluence after plating. Transmission electron microscopy was carried out to monitor the morphological features of cell differentiation. Alkaline Phosphatase (ALP), Aminopeptidase N (APN) and Dipeptidyl Peptidase IV (DPP IV) activity were assayed as known markers of intestinal cell differentiation. The measure of TEER and the apparent permeability of Lucifer Yellow allows to monitor the integrity of the tight junctions and the permeability of the cell layer formed. At T0 a classical monolayer is present, with a mixed population of immature absorptive elements and secretive cells. At T6 and T14, cells are progressively organized in a multilayer with a parallel growth of microvilli. At T6, co-culture demonstrates good properties of permeability and barrier components, such as mucus, representing an appropriate model for absorption study. At T14, the brush border is even more developed respect to T6 and, together with the increase of the specific activity of ALP, APN, and DPP IV, indicate co-culture as a good model for digestion study. The advantage of this co-culture described is the use of the whole cell population without particular inducers of subclones and growth supports. In conclusion, the morphological and biochemical features of co-cultured parental cells change with time, strongly supporting i) an active interaction between the two parental cell lines and ii) the versatility of this model, with more than one prevalent cell type depending on the post confluent stage

Effects of the COVID-19 lockdowns on the management of coral restoration projects

Coral restoration initiatives are gaining significant momentum in a global effort to enhance the recovery of degraded coral reefs. However, the implementation and upkeep of coral nurseries are particularly demanding, so that unforeseen breaks in maintenance operations might jeopardize well-established projects. In the last 2 years, the COVID-19 pandemic has resulted in a temporary yet prolonged abandonment of several coral gardening infrastructures worldwide, including remote localities. Here we provide a first assessment of the potential impacts of monitoring and maintenance breakdown in a suite of coral restoration projects (based on floating rope nurseries) in Colombia, Seychelles, and Maldives. Our study comprises nine nurseries from six locations, hosting a total of 3,554 fragments belonging to three coral genera, that were left unsupervised for a period spanning from 29 to 61 weeks. Floating nursery structures experienced various levels of damage, and total fragment survival spanned from 40 to 95% among projects, with Pocillopora showing the highest survival rate in all locations present. Overall, our study shows that, under certain conditions, abandoned coral nurseries can remain functional for several months without suffering critical failure from biofouling and hydrodynamism. Still, even where gardening infrastructures were only marginally affected, the unavoidable interruptions in data collection have slowed down ongoing project progress, diminishing previous investments and reducing future funding opportunities. These results highlight the need to increase the resilience and self-sufficiency of coral restoration projects, so that the next global lockdown will not further shrink the increasing efforts to prevent coral reefs from disappearing.Peer reviewe