Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.     

Idiosyncratic Drug-Induced Liver Injury: Mechanistic and Clinical Challenges

Idiosyncratic drug-induced liver injury (IDILI) remains a significant problem for patients and drug development. The idiosyncratic nature of IDILI makes mechanistic studies difficult, and little is known of its pathogenesis for certain. Circumstantial evidence suggests that most, but not all, IDILI is caused by reactive metabolites of drugs that are bioactivated by cytochromes P450 and other enzymes in the liver. Additionally, there is overwhelming evidence that most IDILI is mediated by the adaptive immune system; one example being the association of IDILI caused by specific drugs with specific human leukocyte antigen (HLA) haplotypes, and this may in part explain the idiosyncratic nature of these reactions. The T cell receptor repertoire likely also contributes to the idiosyncratic nature. Although most of the liver injury is likely mediated by the adaptive immune system, specifically cytotoxic CD8+ T cells, adaptive immune activation first requires an innate immune response to activate antigen presenting cells and produce cytokines required for T cell proliferation. This innate response is likely caused by either a reactive metabolite or some form of cell stress that is clinically silent but not idiosyncratic. If this is true it would make it possible to study the early steps in the immune response that in some patients can lead to IDILI. Other hypotheses have been proposed, such as mitochondrial injury, inhibition of the bile salt export pump, unfolded protein response, and oxidative stress although, in most cases, it is likely that they are also involved in the initiation of an immune response rather than representing a completely separate mechanism. Using the clinical manifestations of liver injury from a number of examples of IDILI-associated drugs, this review aims to summarize and illustrate these mechanistic hypotheses.     

Different ratios of carbon sources in the fermentation of cheese whey and glucose as substrates for hydrogen and ethanol production in continuous reactors

The fermentation of glucose, cheese whey and the mixture of glucose and cheese whey were evaluated in this study from two inocula sources (sludge from a UASB reactor for swine wastewater treatment and poultry slaughterhouse) for hydrogen production in continuous anaerobic fluidized bed reactors (AFBR). For all fermentations, a hydraulic retention time (HRT) of 6 h and a substrate concentration of 5 g COD L⁻¹ were used. In glucose fermentation, the maximum hydrogen yield (HY) was 1.37 mmol H₂ g⁻¹ COD. The co-fermentation of the cheese whey and glucose mixture was favorable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H₂ g⁻¹ COD and 3.45 mol EtOH g⁻¹ COD in AFBR2. The utilization of cheese whey as a sole substrate resulted in an HY of 1.9 mmol H₂ g⁻¹ COD. Throughout the study, ethanol fermentation was evident.     

The activation of eco-driving mental models: Can text messages prime drivers to use their existing knowledge and skills?

Eco-driving campaigns have traditionally assumed that drivers lack the necessary knowledge and skills and that this is something that needs rectifying. Therefore, many support systems have been designed to closely guide drivers and fine-tune their proficiency. However, research suggests that drivers already possess a substantial amount of the necessary knowledge and skills regarding eco-driving. In previous studies, participants used these effectively when they were explicitly asked to drive fuel-efficiently. In contrast, they used their safe driving skills when they were instructed to drive as they would normally. Hence, it is assumed that many drivers choose not to engage purposefully in eco-driving in their everyday lives. The aim of the current study was to investigate the effect of simple, periodic text messages (nine messages in 2 weeks) on drivers’ eco- and safe driving performance. It was hypothesised that provision of eco-driving primes and advice would encourage the activation of their eco-driving mental models and that comparable safety primes increase driving safety. For this purpose, a driving simulator experiment was conducted. All participants performed a pre-test drive and were then randomly divided into four groups, which received different interventions. For a period of 2 weeks, one group received text messages with eco-driving primes and another group received safety primes. A third group received advice messages on how to eco-drive. The fourth group were instructed by the experimenter to drive fuel-efficiently, immediately before driving, with no text message intervention. A post-test drive measured behavioural changes in scenarios deemed relevant to eco- and safe driving. The results suggest that the eco-driving prime and advice text messages did not have the desired effect. In comparison, asking drivers to drive fuel-efficiently led to eco-driving behaviours. These outcomes demonstrate the difficulty in changing ingrained habits. Future research is needed to strengthen such messages or activate existing knowledge and skills in other ways, so driver behaviour can be changed in cost-efficient ways.     

Design characteristics of a workload manager to aid drivers in safety–critical situations

Cognition, Technology & Work - The objective of this study was to evaluate a workload manager designed to supervise the presentation of in-vehicle information for two age groups of drivers...     

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s⁻¹). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A₂ (TxA₂), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.     

Broad Spectrum Anti-Bacterial Activity and Non-Selective Toxicity of Gum Arabic Silver Nanoparticles

Silver nanoparticles (AgNPs) are the most commercialized nanomaterials and presumed to be biocompatible based on the biological effects of the bulk material. However, their physico-chemical properties differ significantly to the bulk materials and are associated with unique biological properties. The study investigated the antimicrobial and cytotoxicity effects of AgNPs synthesized using gum arabic (GA), sodium borohydride (NaBH₄), and their combination as reducing agents. The AgNPs were characterized using ultraviolet-visible spectrophotometry (UV-Vis), dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR). The anti-bacterial activity was assessed using agar well diffusion and microdilution assays, and the cytotoxicity effects on Caco-2, HT-29 and KMST-6 cells using MTT assay. The GA-synthesized AgNPs (GA-AgNPs) demonstrated higher bactericidal activity against all bacteria, and non-selective cytotoxicity towards normal and cancer cells. AgNPs reduced by NaBH₄ (C-AgNPs) and the combination of GA and NaBH₄ (GAC-AgNPs) had insignificant anti-bacterial activity and cytotoxicity at ≥50 µg/mL. The study showed that despite the notion that AgNPs are safe and biocompatible, their toxicity cannot be overruled and that their toxicity can be channeled by using biocompatible polymers, thereby providing a therapeutic window at concentrations that are least harmful to mammalian cells but toxic to bacteria.     

Comparison of two low-hazard organic solvents as individual and cosolvents for the fabrication of polysulfone membranes

Petroleum-derived solvents commonly used in membrane fabrication are often hazardous and toxic, so the investigation of safer alternatives is important. In this study, two low-hazard solvents, methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate (Rhodiasolv® PolarClean) and gamma-valerolactone (GVL), were investigated as sole solvents and as cosolvents to cast polysulfone membranes via nonsolvent induced phase inversion. Normalized viscosity was introduced as an indicator of dope solution homogeneity and was used to compare the required time of mixing to achieve full dissolution of the polymer in the different solvents/solvent mixtures. All dope solutions made with low-hazard solvents were found to be more viscous than those made with traditional solvents, which meant additional mixing time was needed, and that fabricated membranes were morphologically different. With respect to operation, membranes cast from dope solutions containing equal amounts of PolarClean and GVL displayed the most similar flux curves and solute rejection to those made using the traditional solvent tested.