APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.     

The Somma–Vesuvius complex and the Phlaegrean Fields caldera: New chronological data of several eruptions of the Copper–Middle Bronze Age period

Radiocarbon dating of short-lived sample materials is a useful tool applied to date deposits of volcanic eruptions. Several archaeological sites discovered and excavated in Campania witnessed important volcanic eruptions, which occurred in the Copper and Middle Bronze Ages. These eruptions come from the Somma–Vesuvius complex and the Phlaegrean Fields caldera. At least four Plinian eruptions have been identified in the eruptive history of Somma–Vesuvius, interspersed by interplinian events, called protohistoric, which occurred between Avellino and Pompeii. At S. Paolo Belsito a stratigraphic sequence below Avellino and above the first two protohistoric events after Avellino were highlighted; while Nola (Naples) gives new information on the chronology of Avellino. Sites like Caivano and Gricignano D’Aversa, involved by the Agnano 3, Paleoastroni 2 and Agnano Monte Spina eruptions were highlighted and investigated. In this work, we want to clarify the chronology of some eruptions by comparing our results with previous data. Charcoal, bone and seed samples were collected, treated and measured at the CIRCE laboratory in Caserta.     

Pressure‐Tunable Ambipolar Conduction and Hysteresis in Thin Palladium Diselenide Field Effect Transistors

Few‐layer palladium diselenide (PdSe₂) field effect transistors are studied under external stimuli such as electrical and optical fields, electron irradiation, and gas pressure. The ambipolar conduction and hysteresis are observed in the transfer curves of the as‐exfoliated and unprotected PdSe₂ material. The ambipolar conduction and its hysteretic behavior in the air and pure nitrogen environments are tuned. The prevailing p‐type transport observed at atmospheric pressure is reversibly turned into a dominant n‐type conduction by reducing the pressure, which can simultaneously suppress the hysteresis. The pressure control can be exploited to symmetrize and stabilize the transfer characteristics of the device as required in high‐performance logic circuits. The transistors are affected by trap states with characteristic times in the order of minutes. The channel conductance, dramatically reduced by the electron irradiation during scanning electron microscope imaging, is restored after an annealing of several minutes at room temperature. The work paves the way toward the exploitation of PdSe₂ in electronic devices by providing an experiment‐based and deep understanding of charge transport in PdSe₂ transistors subjected to electrical stress and other external agents.     

Limnological features of Funil Reservoir (R.J., Brazil) and indicator properties of rotifers and cladocerans of the zooplankton community

Funil Reservoir receives inflow from a highly industrialized region and acts as a natural sink to pollutants. Among the consequences of the uncontrolled nutrient loading is the constant presence and periodic heavy blooms of Microcystis aeruginosa . This study verified limnological features and zooplankton assemblage of the Funil Reservoir focusing on the environmental-indicator properties of rotifers and cladocerans. The summer bloom of M. aeruginosa caused reductions in water transparency, nitrate and orthophosphate concentrations and raises in chlorophyll a , pH, dissolved oxygen and chemical oxygen demand values. All zooplankton species presented spatial and temporal variations with the exception of the copepods, which were present in all samples. According to canonical correspondence analysis, ammonium was the variable most related to zooplankton variability and different Rotifera and Cladocera assemblages indicated distinct environment conditions. Rotifer taxa associated with increases of water temperature and chlorophyll a concentration were found with high densities during M. aeruginosa blooms. Species of rotifer and cladocerans are suggested as indicators that can be used to identify different physical and chemical gradients or eutrophic increases in Funil Reservoir.     

Shortening of Free-running XeCl Laser Pulses by Stimulated Brillouin Scattering

Abstract

A stimulated Brillouin scattering mirror has been used to ‘temporally tune’ from nanoseconds to subnanoseconds the width of laser pulses generated by a free running XeCl oscillator, equipped with a super-Gaussian reflectivity unstable resonator. Pulse shortening techniques by stimulated Brillouin scattering (SBS) and by truncated SBS have been investigated. A maximum pulse shortening of about fifty has been achieved by truncated SBS.     

Semi-quantitative monitoring of confluence of adherent mesenchymal stromal cells on calcium-phosphate granules by using widefield microscopy images

The analysis of cell confluence and proliferation is essential to design biomaterials and scaffolds to use as bone substitutes in clinical applications. Accordingly, several approaches have been proposed in the literature to estimate the area of the scaffold covered by cells. Nevertheless, most of the approaches rely on sophisticated equipment not employed for routine analyses, while the rest of them usually do not provide significant statistics about the cell distribution. This research aims at studying confluence and proliferation of mesenchymal stromal cells (MSC) adherent on OSPROLIFE^®, a commercial biomaterial in the form of granules. In particular, we propose a Computer Vision approach that can routinely be employed to monitor the surface of the single granules covered by cells because only a standard widefield fluorescent microscope is required. In order to acquire significant statistics data, we analyse wide-area images built by using MicroMos v2.0, an updated version of a previously published software specific for stitching brightfield and phase-contrast images manually acquired via a widefield microscope. In particular, MicroMos v2.0 permits to build accurate “mosaics” of fluorescent images, after correcting vignetting and photo-bleaching effects, providing a consistent representation of a sample region containing numerous granules. Then, our method allows to make automatically a statistically significant estimate of the percentage of the area of the single granules covered by cells. Finally, by analysing hundreds of granules at different time intervals we also obtained reliable data regarding cell proliferation, confirming that not only MSC adhere onto the OSPROLIFE^® granules, but even proliferate over time.     

A heat-flux based “building block” approach for solving heat conduction problems

A numerical approximation of the Green’s function equation based on a heat-flux formulation is given. It is derived by assuming as a functional form of the surface heat flux a stepwise variation with space and time. The obtained approximation is very important in investigation of the inverse heat conduction problems (IHCPs) because it gives a convenient expression for the temperature in terms of the heat flux components. Additionally, it is very important for the unsteady surface element (USE) method which is a modern boundary discretization method. Green’s function approximate solution equation (GFASE) also creates ‘naturally’ fixed groups or modules of work elements called “building blocks” that may be added together to obtain space and time values of temperature. In the current case, they are subject to a partial heating by an applied surface heat flux. The “building block” solution can be derived by using the various analytical and numerical approaches available in heat conduction literature though the exact analysis is preferable, as discussed in the text. Poorly-convergent series deriving from Green’s functions approach are replaced by closed-form algebraic solutions.     

Linux real-time framework for fusion devices

A new framework for the development and execution of real-time codes is currently being developed and commissioned at JET. The foundations of the system are Linux, the Real Time Application Interface (RTAI) and a wise exploitation of the new i386 multi-core processors technology.The driving motivation was the need to find a real-time operating system for the i386 platform able to satisfy JET Vertical Stabilisation Enhancement project requirements: 50 μs cycle time. Even if the initial choice was the VxWorks operating system, it was decided to explore an open source alternative, mostly because of the costs involved in the commercial product.The work started with the definition of a precise set of requirements and milestones to achieve: Linux distribution and kernel versions to be used for the real-time operating system; complete characterization of the Linux/RTAI real-time capabilities; exploitation of the multi-core technology; implementation of all the required and missing features; commissioning of the system.Latency and jitter measurements were compared for Linux and RTAI in both user and kernel-space. The best results were attained using the RTAI kernel solution where the time to reschedule a real-time task after an external interrupt is of 2.35 ± 0.35 μs. In order to run the real-time codes in the kernel-space, a solution to provide user-space functionalities to the kernel modules had to be designed. This novel work provided the most common functions from the standard C library and transparent interaction with files and sockets to the kernel real-time modules. Kernel C++ support was also tested, further developed and integrated in the framework.The work has produced very convincing results so far: complete isolation of the processors assigned to real-time from the Linux non real-time activities, high level of stability over several days of benchmarking operations and values well below 3 μs for task rescheduling after external interrupt. From being the alternative option, RTAI has been finally chosen as the platform for the project. A first stable version of the framework has been integrated on the JET system and is already being commissioned. It will be soon be used on the Vertical Stabilisation Enhancement for the Plasma Control Upgrade (PCU) project at JET.     

Tool-supported requirements prioritization: Comparing the AHP and CBRank methods

Requirements prioritization aims at identifying the most important requirements for a software system, a crucial step when planning for system releases and deciding which requirements to implement in each release. Several prioritization methods and supporting tools have been proposed so far. How to evaluate their properties, with the aim of supporting the selection of the most appropriate method for a specific project, is considered a relevant question.In this paper, we present an empirical study aiming at evaluating two state-of-the art tool-supported requirements prioritization methods, AHP and CBRank. We focus on three measures: the ease of use, the time-consumption and the accuracy. The experiment has been conducted with 23 experienced subjects on a set of 20 requirements from a real project. Results indicate that for the first two characteristics CBRank overcomes AHP, while for the accuracy AHP performs better than CBRank, even if the resulting ranks from the two methods are very similar. The majority of the users found CBRank the “overall best” method.